Antidiabetic compounds

ABSTRACT

The present invention provides novel peptidomimetics, of formula (I), which primarily act as glucose dependent insulin secretagogues. Furthermore, it was found that these peptidomimetics showed glucagon receptor antagonistic activity, along with the GLP-1 receptor agonistic activity.
 
A-Z 1 —Z 2 —Z 3 —Z 4 —Z 5 —Z 6 —Z 7 —Z 8 —Z 9 —Z 10 —Z 11 —B  (I)

FIELD OF INVENTION

The present invention relates to novel compounds of general formula (I),their tautomeric forms, their pharmaceutically acceptable salts andpharmaceutical compositions containing them:A-Z₁—Z₂—Z₃—Z₄—Z₅—Z₆—Z₇—Z₈—Z₉—Z₁₀—Z₁₁—B  (I)

The present invention also relates to a processes for preparingcompounds of general formula (I), their tautomeric forms, theirpharmaceutically acceptable salts and pharmaceutical compositionscontaining them.

BACKGROUND TO THE INVENTION

Diabetes is characterized by impaired insulin secretion from pancreaticβ-cells, insulin resistance or both (Cavaghan, M. K., et al., J. Clin.Invest. 2000, 106, 329). Majority of type 2 diabetic patients can betreated with agents that reduces hepatic glucose production (glucagonantagonist), reduce glucose absorption form GIT, stimulate β-cellfunction (insulin secretagogues) or with agents that enhance the tissuesensitivity of the patients towards insulin (insulin sensitizes). Thedrugs presently used to treat type 2 diabetes include α-glucosidaseinhibitors, insulin sensitizers, insulin secretagogues and K_(ATP)channel blocker (Chehade, J. M., et al., Drugs, 2000, 60, 95). However,almost one-half of type 2 diabetic subjects lose their response to theseagents, over a period of time and thereby require insulin therapy.Insulin treatment has several drawbacks, it is injectable, produceshypoglycemia and causes weight gain (Burge, M. R., Diabetes Obes.Metab., 1999, 1, 199).

Problems with the current treatment necessiate new therapies to treattype 2 diabetes. In this regard, glucagon-like peptide-1 (GLP-1)agonist, which promote glucose-dependent insulin secretion in thepancreas and glucagon receptor antagonist, which inhibit hepatic glucoseproduction by inhibiting glycogenolysis and gluconeogenesis, were foundto be therapeutically potential. Thus GLP-1 agonist and glucagonantagonist together were found to reduce the circulating glucose levelsand represent useful therapeutic agents for the treatment and preventionof type 2 diabetes (Perry, T. A., et al., Trends Pharmacol. Sci., 2003,24, 377).

Glucagon and GLP-1 are members of structurally related peptide hormonefamily (secretin family). Glucagon and GLP-1 constitute a highlyhomologous set of peptides because these two hormones originate from acommon precursor, preproglucagon, which upon tissue-specific processingleads to production of GLP-1 predominantly in the intestine and glucagonin the pancreas (Jiang, G., et al., Am. J. Physiol. Endocrinol. Metab.,2003, 284, E671-678). The receptors for these two peptides arehomologous (58% identity) and belong to the class B family of G-proteincoupled receptors (GPCRs). Class-B GPCRS is also called as the secretinreceptor family, which consist of 15 peptide-binding receptors inhumans. GPCR receptors comprise an extracellular N-teiininal domain of100-160 residues, connected to a juxtamembrane domain (J-domain) ofseven membrane-spanning α-helices with intervening loops and a.C-terminal tail (Brubaker, P. L., et al., Receptors Channels, 2002, 8,179). Class B GPCRs are activated by endogenous peptide ligands ofintermediate size, typically 30-40 amino acids (Hoare, S. R. J., Drug.Discovery Today, 2005, 10, 423; Gether, U., Endocrine Reviews, 2000, 21,90).

Glucagon is a 29-amino acid peptide hormone processed from proglucagonin pancreatic α-cells by PC2. Glucagon acts via a seven transmembraneGPCRs, consisting of 485 amino acids. Glucagon is released into thebloodstream when circulating glucose is low. The main physiological roleof glucagon is to stimulate hepatic glucose output, thereby leading toincrease in glycemia (Tan, K., et al., Diabetologia, 1985, 28, 435).Glucagon provides the major counterregulatory mechanism for insulin inmaintaining glucose homeostasis in vivo. Glucagon and its receptorrepresent potential targets for the treatment of diabetes. Antagonisingglucagon action by blocking the action of the secreted glucagon atglucagon receptor (glucagon antagonist) or by inhibiting (suppressing)the glucagon production itself represents a new avenue for interventionof diabetes and metabolic disorders (Unson, C. G., et al., Peptides,1989, 10, 1171; Parker, J. C., Diabetes, 2000, 49, 2079; Johnson, D. G.,Science, 1982, 215, 1115).

The GLP-1 (7-36) amide is a product of the preproglucagon gene, which issecreted from intestinal L-cells, in response to the ingestion of food.The physiological action of GLP-1 has gained considerable interest.GLP-1 exerts multiple action by stimulating insulin secretion frompancreatic β-cells, in a glucose dependent manner (insulinotropicaction). GLP-1 lowers circulating plasma glucagon concentration, byinhibiting its secretion (production) from α-cells (Drucker D. J.,Endocrinology, 2001, 142, 521-527). GLP-1 also exhibits properties likestimulation of β-cell growth, appetite suppression, delayed gastricemptying and stimulation of insulin sensitivity (Nauck, M. A., Horm.Metab. Res., 2004, 36, 852). Currently, various analogs of GLP-1 andEX-4, such as Liraglutide/NN2211 (Novo Nordisk; Phase-III; WO 1998008871), BIM 51077 (Ipsen; Phase-II; WO 2000 034331), CJC-1131(ConjuChem; Phase-II; WO 2000 069911) and ZP-10 (Zealand & Aventis;Phase-II; WO 2001 004156) are in different stages of clinicaldevelopment (Nauck M. A., Regulatory Peptides, 2004, 115, 13). Recently,BYETTA® (Exendin-4, AC 2933; U.S. Pat. No. 5,424,286), has been launchedin the US market (Amylin & Lilly). However, all the existing GLP-1agonists are delivered by the parenteral route of administration, so thepatient incompliance is major problem with the existing GLP-1 basedtherapy.

The effector system of glucagon and GLP-1 receptors is the AdenylylCyclase (AC) enzyme. Interaction of glucagon or GLP-1 agonist withglucagon or GLP-1 receptors (GLP-1 R) respectively causes activation ofAC, which converts ATP to cAMP. Increase in the intracellular cAMP levelraises the ratio of ADP/ATP, thereby initiating the cell depolarization(due to closure of K_(ATP) channel). Increase in the intracellular cAMPlevel also activates Protein Kinase (PK-A & PK-C), which raises thecystolic Ca²⁺ concentration, by opening of L-type of Ca²⁺ channel. Anincrease in the intracellular Ca²⁺ leads to exocytosis of insulin, inpancreatic β-cells and glucagon peptide in α-cells (Fehmann, H. C.,Endocr. Rev., 1995, 16, 390).

GLP-1 and glucagon sequences alignment shown below represent the primarystructural relationships:

Glucagon: (Seq. ID No: 1) NH₂-¹ HSQGTFTSD ⁹YSKYLDSRRAQDFVQWLMNT-CONH₂GLP-1(7-36): (Seq. ID No: 2) NH₂-¹ HAEGTFTSD⁹VSSYLEGQAAKEFIAWLVKGR-CONH₂First N-terminal 1-9 residues of GLP-1 peptide, with C-terminal amide:

NH₃ ⁽⁺⁾-¹ HAE ⁽⁻⁾ GTFTSD ⁹⁽⁻⁾-CONH₂ (Seq. ID No: 3): Net charge NegativeFirst N-terminal 1-9 residues of Glucagon peptide, with C-terminalamide:

NH₃ ⁽⁺⁾-¹ HSQGTFTSD ⁹⁽⁻⁾-CONH₂ (Seq. ID No: 4): Net charge NeutralSingle-letter abbreviations for amino acids can be found in Zubay, G.,Biochemistry 2^(nd) ed., 1988, MacMillan Publishing, New York, p. 33.

Native or synthetic GLP-1 peptides are rapidly metabolized by theproteolytic enzymes, such as dipeptidyl peptidase-IV (DPP-IV) into aninactive metabolite, thereby limiting the use of GLP-1 as a drug(Deacon, C. F., Regulatory Peptides, 2005, 128, 117). Similarly, severalnonpeptidyl and peptidyl glucagon receptor antagonist of diversestructures have been reported over recent years, but none of them are inactive development or under clinical trials (Kurukulasuriya, R., ExpertOpinion Therapeutic Patents, 2005, 15, 1739; Lau, J., J. Med. Chem.,2007, 50, 113; Petersen, K. F. Diabetologia, 2001, 44, 2018; Cascieri,M. A., JBC, 1999, 274, 8694). It is believed that identifying nonpeptideligands (especially agonist) for class B GPCRs is the principlebottleneck in drug discovery. HTS has apparently yielded few hits (US2005/6927214; WO 2000/042026; US 2007/0043093), however, screening ofthose hits against corresponding receptors, especially under in vivocondition (animal models) prone to be false negatives (Murphy, K. G.,PNAS, 2007, 104, 689).

Glucagon and GLP-1 both play major roles in overall glucose homeostasis(Drucker, D. J., J. Clin. Invest., 2007, 117, 24; Bollyky, J., J. Clin.Endocrinol. Metab., 2007, 92, 2879). Glucagon increases plasma glucoseconcentrations by stimulating gluconeogenesis and glycogenolysis in theliver while GLP-1 lowers plasma glucose concentrations mediated byglucose dependent insulin secretion (Mojsov, S., et al., JBC., 1990,265, 8001). Knowing the importance of both glucagon peptide and GLP-1 inmaintaining normal blood glucose concentrations, in the recent years,there has been considerable interest in identifying a single ligand,which act as glucagon receptor antagonists and GLP-1 receptor agonists(Claus, T. H., J. Endocrinology, 2007, 192, 371; Pan C. Q., JBC, 2006,281, 12506).

Although identification of potent nonpeptide GLP-1 agonist may bedifficult (Chen, D., PNAS, 2007, 104, 943; Knudsen, L. B., PNAS, 2007,104, 937) but the design of a hybrid peptidomimetic acting as bothglucagon antagonist and GLP-1 receptor agonist would likely to provide anovel approach for the treatment of type 2 diabetes (Claus, T B., J.Endocrinology, 2007, 192, 371). Structure-activity relationship (SAR)studies have been reported in the literature to determine the role ofindividual amino acids in both the glucagon and GLP-1 sequences (Runge,S., JBC, 2003, 278, 28005; Mann, R., Biochem. Soc. Trans., 2007, 35,713). Glucagon and GLP-1 have no defined structure in aqueous solution,but in the presence of micelles or in the membrane mimetic environment,they adopt an alpha; helical structure in the mid-section, with flexibleN- and C-terminal regions (Thornton, K., Biochemistry, 1994, 33, 3532;Neidigh, J. W., Biochemistry, 2001, 40, 13188). This suggests that thehelical structure is required for binding of peptide ligands to theirrespective receptors. Mutations or deletion of amino acids in theN-terminal region of both the peptides results in receptor antagonistsor inactive compounds, suggesting the importance of the N-terminus forreceptor activation by both the glucagon and GLP-1 peptides (Hjorth, S.A., JBC., 1994, 269, 30121; Green, B. D., J. Mol. Endocrinology, 2003,31, 529). In vivo, GLP-1 gets rapidly degraded by dipeptidyl-peptidaseIV (DPP IV), a protease responsible for cleaving peptides containingproline or alanine residues in the penultimate. N-terminal position,resulting in the inactive metabolites. Substitution of the DPP-IVsusceptible sites, such as substitution of Ala at 2^(nd) position ofGLP-1 peptide with D-Ala, Aib, greatly improves plasma stability(Deacon, C. F., Diabetes, 1998, 47, 764).

In the present investigation, we found that coupling of N-terminalsequence of glucagon peptide (first 1-9 residues, Seq. ID. No. 4) with adipeptide of two unnatural amino acids resulted in the identification ofnovel class of peptidomimetics having both the glucagon antagonistic andGLP-1 agonistic activities, at varying degree of selectivity. To enhancethe duration of action and stability against DPP-IV enzyme, we havesite-specifically modified the hybrid peptidomimetics selectively atposition Z₂ with unnatural amino acids such as D-Ala, Aib and1-amino-cyclopropane carboxylic acid (ACP) and succeeded in identifyingshort peptidomimetics. Some of the peptidomimetics showed efficacy evenby oral route of administration, while retaining both the glucagonantagonistic and GLP-1 agonistic activities.

PRIOR ART

A series of human GLP-1 mimics, have been reported with general formulaXaa1-Xaa11, wherein Xaa1-Xaa9 represent the first 1-9 residues of GLP-1peptide (HAEGTFTSD; Seq. ID No. 3), with some analogs wherein Xaa2represents either Ala Or are optionally replaced with Aib, Xaa3represents amino acids with carboxylic acid side chain such as glutamicacid, aspartic acid etc. but not the Gln (Q), which is conserved inN-terminal sequence of Glucagon peptide (HSQGTFTSD, Seq. ID No. 4). Xaa6represents Phe or are optionally replaced with -α-Me-2F-Phe-, Xaa9represent amino acids with carboxylic acid or amide side chains such asaspartic acid, glutamic acid, asparagine etc., Xaa10 & Xaa11 representscombination of substituted or unsubstituted biphenyl alanine (Bip)derivatives (WO 2003/033671A2; US 2004/0127423 A1; WO 2004/094461 A2; US2006/0004222 A1; WO 2006/014287 A1; WO 2006/127948 A2; WO 2007/082264A2; US 2007/0021346 A1; US2007/0099835).

The present invention provides novel peptidomimetics of formula (I)(hereinafter referred to as peptidomimetics), which primarily act as aglucagon receptor antagonist and also exhibit GLP-1R agonistic effects.Different peptidomimetics reported in this invention showed significantglucose dependent insulin secretion (in vitro) and reduce circulatingglucose levels (in vivo), with different level of affinity/selectivitytowards glucagon and GLP-1 receptors. Furthermore, these peptidomimeticsshowed increased stability to proteolytic cleavage, especially againstDPP-IV enzyme with improved half-life. Some of the peptidomimetics werefound to be stable against GIT enzymes and acidic pH of stomach, withoral bioavailability, making them suitable candidate for thetreatment/mitigation/prophylaxis of both type 1 & type 2 diabetes,metabolic disorders and related disorders.

Design Strategy for the Dual Acting Peptidomimetics (Glucagon Antagonistand GLP-1 Agonist):

The similarity between glucagon and GLP-1 peptides and also betweentheir respective receptors raises the possibility of producing hybridpeptidomimetics that can bind with both the receptors, but selectivelyexhibit antagonistic activity at glucagon receptor and agonisticactivity at GLP-1 receptor. Furthermore, in order to have oralbioavailibility and increased metabolic stability, it is also essentialto develop peptidomimetics, with shorter amino acid sequence.

A general mechanism of peptide ligand interaction with class B GPCRs hasemerged and is termed as the ‘two-domain’ model. The C-terminal portionof the peptide binds the N-domain of the receptor, confirm binding ofligand with the receptor and the N-terminal ligand region binds theJ-domain, an interaction that activates the receptor and stimulatesintracellular signaling, (FIG. 1) (Ji, T. H., JBC, 1988, 273, 17299;Hjorth, S. A., et al., Regulatory Peptides, 1996, 64, 70). Thus theN-domain of GLP-1 and glucagon receptors determine the selectivity ofthe C-terminal portion of GLP-1 and glucagon peptides respectively(Hoare, S. R. J., Drug. Discovery Today, 2005, 10, 423).

In order to design short chain peptides/peptidomimetics that binds withboth receptors and exhibits agonist activity on the GLP-1 receptor butantagonist activity on the glucagon receptor, we decided to exploretwo-domain model concept (reported in the literature for Class-B,GPCRs), which indicates importance of both the activation and bindingcomponents of endogenous peptide ligands.

Recently, series of chimeric peptides (prepared recombinantly) has beenreported, which act as both GLP-1 receptor agonist and glucagon receptorantagonist, constructed mainly by combining the N-terminal residues ofglucagon peptide (residues 1-26) with last C-terminal 4 residues ofGLP-1 peptide (VKGR) (Pan C. Q., et al., U.S. Pat. No. 6,864,069 B2; PanC. Q., JBC, 2006, 281, 12506). However, these reports describefull-length chimeric peptides exhibiting GLP-1 receptor agonist andglucagon receptor antagonist properties and not the short chainpeptides/peptidomimetics. In broad sense, this concept indicates thatincorporation of C-terminal sequence of GLP-1 (especially last 4residues, VKGR) into glucagon sequence lead to the formation of novelpeptides, which act as both GLP-1 receptor agonist and glucagon receptorantagonist.

Thus for dual GLP-1 agonist activity and glucagon antagonistic activity,binding component of GLP-1 peptide sequence is essential (atleast last 4residues) and for activation of GLP-1 receptor and inhibition ofglucagon receptor, N-terminal sequence of glucagon peptide is required.However, while designing such dual acting peptidomimetics, it is alsoessential to keep shortest peptide sequence so that novel peptidomimeticcould be orally bioavailable. Furthermore, to improve metabolicstability against DPP-IV enzyme, which selectively cleave N-terminaldipeptide at the penultimate N-terminal position, it is essential toincorporate DPP-IV stable amino acid, especially at 2^(nd) position.

In search of short length binding component, we explored the GLP-1 andglucagon receptor binding affinities of substituted or unsubstitutedunnatural dipeptide amino acids (Bip-Bip). Such unnatural dipeptideamino acids have been reported, in combination of first 1-9 N-terminalresidues of GLP-1 peptide sequence (WO 2003/033671A2; US 2004/0127423A1; WO 2004/094461 A2; US 2006/0004222 A1; WO 2006/014287 A1; WO2006/127948. A2; WO 2007/082264 A2; US 2007/0021346 A1; US2007/0099835)as potential peptidomimetics with GLP-1 agonist activity. Surprisingly,in our in vitro human GLP-1 and Glucagon receptors assays, dipeptides(Bip(OMe)-Bip(2Me)/Bip(OMe)-Bip(Pyr)/Bip(OMe)-Bip(2F)/Bip(OMe)-Bip(2CF₃)),[FIG. 2], showed antagonistic activity both in human GLP-1 and Glucagonreceptors assay, [FIG. 3].

Knowing the binding affinity of dipeptide towards both the GLP-1 andglucagon receptors, we decided to couple this binding component with theactivation unit. Surprisingly, instead of first 9 residues of N-terminalsequence of GLP-1 peptide (HAEGTFTSD; Seq. ID No. 3), when we attachedthis dipeptide to first 9 residues of N-terminal sequence of Glucagonpeptide (¹HSQGTFTSD⁹; Seq. ID No: 4), we found that this peptidomimetic(NH₃ ⁺-HSQGTFTSD-Bip(OMe)-Bip(2Me)-CONH₂; Seq. ID No. 5) primarilyshowed glucagon receptor antagonistic activity, along with the GLP-1receptor agonistic activity, [FIG. 4].

Furthermore, to increase the stability of this dual actingpeptidomimetic against proteolytic cleavage, especially against DPP-IV(Dipeptidyl peptidase-IV) enzyme, we have site-specifically modified thehybrid peptidomimetic, selectively at position-2, with unnatural aminoacids such as D-Ala, Aib or ACP and succeeded in identifyingmetabolically stable short peptidomimetics, while retaining both theGLP-1 agonist and glucagon antagonist activities.

Oral route of drug administration is universally accepted by patientsdue to its ease of administration but it encounters absorption andenzymatic barriers along with the first-pass effect. Although, the oralroute of drug administration is the route of choice, the delivery ofpeptide and protein drugs by this route is currently limited due totheir poor permeability and rapid degradetion in the gastrointestinaltract. Therefore, most of the commercially available peptide and proteindrugs are administered by the parenteral route. However, due to patientnon-compliance and short half-life of peptide and protein drugs, theparenteral route of administration is not suitable for the delivery ofthese drugs. Thus, the clinical utilities of most of peptide and proteindrugs are currently limited due to their unfavorable physiochemicalproperties, such as high molecular weight, metabolic susceptibility andhydrophilicity (Morishita, M., Drug Discovery Today, 2006, 11, 905).

Epithelia represent a good target for the delivery ofbiopharmaceuticals. However, semi-permeable nature of epithelia limitthe passage of peptide and protein drugs through their surfaces andthereby act as a principal absorption barrier (Arhewon, I. M., AfricanJ. Biotechnology, 2005, 4, 1591). The cell membrane of epithelial cellsis made up of a continuous bilayer layer of membrane lipids andproteins. The membrane lipids consist of amphiphilic, phospholipids;such as phosphatidylcholine, sphingomyelin, phosphatidylserine andphosphatidylethanolamine, along with glycolipids and cholesterolmolecules [FIG. 5].

Phosphatidylcholine and sphingomyelin are neutral phospholipids, whereasphosphatidylserine are negatively charged phospholipid. At physiologicalpH, most epithelia exhibit net negative charge due to the presence ofphosphatidylserine. Thus while designing our peptidomimetics, attemptswere made to avoid net negative charge. In general, in all the sequencesdesigned in this invention, net neutral charge was maintained, whichcould be possible by incorporation first 1-9 residues of N-terminalsequence of glucagon peptide (Seq. ID No. 4) and not the first 1-9residues of N-terminal sequence of GLP-1 peptide (Seq. ID No. 4), whichmainly differ in their net charges (neutral to net negative) due toglutamine (Q; conserved in glucagon sequence) vs glutamic acid (E;conserved in GLP-1 sequence), at position-3. Overall, considerablepotential exists in a single therapeutic compound, functioning as both aGLP-1R agonist and a glucagon receptor antagonist and preferably due toshort peptide chain length, such peptidomimetic are likely to be orallybioavailable for the treatment or prevention of diabetes and relatedmetabolic disorders.

SUMMARY OF THE INVENTION

The present invention describes a group of novel peptidomimetics thatfunction both as an antagonist of the glucagon receptor and agonist ofthe GLP-1 receptor, having different degree of affinity/selectivitytowards both the receptors and useful for reducing circulating glucoselevels and for the treatment of diabetes. These peptidomimetics aredefined by the general formula (I) as given below. The peptidomimeticsof the present invention are useful in the treatment of the human oranimal body, by regulation of insulin and glucagon action. Thepeptidomimetics of this invention are therefore suitable for thetreatment/mitigation/regulation or prophylaxis of type 1 and type 2diabetes and associated metabolic disorders.

PREFERRED EMBODIMENTS

A preferred embodiment of the present invention is to provide novelpeptidomimetics of general formula (I), their tautomeric forms, novelintermediates involved in their synthesis, their pharmaceuticallyacceptable salts, their pharmaceutically acceptable solvates andpharmaceutical compositions containing them or their mixtures, suitablefor the treatment treatment/mitigation/regulation of diabetes.

In another preferred embodiment is provided a process for thepreparation of novel peptidomimetics of general formula (I), theirtautomeric forms, their pharmaceutically acceptable salts,pharmaceutically acceptable solvates and pharmaceutical compositionscontaining them.

In a further preferred embodiment, is provided pharmaceuticalcompositions containing peptidomimetics of general formula (I), theirtautomeric forms, their pharmaceutically acceptable salts, solvates andtheir mixtures having pharmaceutically acceptable carriers, solvents,diluents, excipients and other media normally employed in theirmanufacture.

In a still further preferred embodiment is provided the use of the novelpeptidomimetics of the present invention as antidiabetic agents, byadministering a therapeutically effective & non-toxic amount of thepeptidomimetics of formula (I), or their pharmaceutically acceptablecompositions to the mammals those are in need of such treatment.

ABBREVIATIONS USED

The following abbreviations are employed in the examples and elsewhereherein:

-   Aib=α-Aminoisobutyric acid,-   ACN=Acetonitrile,-   Bip=Biphenylalanine residue,-   Bn=Benzyl,-   Boc=tert-Butoxycarbonyl,-   Bu^(t)=O-tert-butyl group,-   cAMP=Adenosine 3′,5′-cyclic monophosphate,-   DCM=Dichloromethane,-   DMF=N,N-Dimethylformamide,-   DIPCDI=Di-isopropylcarbodiimide,-   DIPEA=Diisopropylethylamine,-   Et=Ethyl,-   Et₂O=Diethyl ether,-   Fmoc=Fluorenylmethoxycarbonyl,-   g=Gram(s),-   GLP-1R=Glucagon Like Peptide-1 Receptor,-   Glucagon R=Glucagon receptor,-   h=Hour(s),-   HOBt=Hydroxybenzotriazole,-   HOAt=7-Aza-hydroxybenzotriazole,-   HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl aminium    hexafluorophosphate;-   HPLC=High Performance Liquid Chromatography,-   i.p.=intraperitonial,-   L=Liter,-   LC/MS=Liquid Chromatography/Mass Spectrometry,-   Me=Methyl,-   Min=minute (s),-   mL=milliliter,-   μl=microliter,-   mg=milligram (s),-   mmol=millimole (s),-   MS=Mass Spectrometry,-   PyBOP=Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate,-   SPPS=Solid Phase Peptide Synthesis,-   sc=subcutaneous,-   TMS=Trimethylsilyl,-   TIPS=Triisopropylsilane,-   TFA=Trifluoroacetic acid,-   TBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium    tetrafluoroborate,-   Trt=Trityl group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates two-domain model for interaction of peptide ligandswith class B GPCRs.

FIG. 2 illustrates structures of dipeptides which showed in vitroGlucagon/GLP-1 antagonistic activity.

FIG. 3 illustrates in vitro Human Glucagon and GLP-1 receptorantagonistic activity of dipeptide (Bip(OMe)-Bip(2Me)).

FIG. 4 illustrates in vitro Human Glucagon receptor antagonisticactivity and GLP-1 receptor agonistic activity with Seq. ID. 5.

FIG. 5 illustrates some of the structural components of epithelialmembrane.

FIG. 6 illustrates examples of orthogonally protected amino acids usedin Fmoc based-solid phase peptide synthesis (SPPS) of peptidomimetics.

FIG. 7 illustrates in vitro DRC and EC₅₀ determination of Exendin(Figure A) and Seq. ID No. 32 (Figure B), in H GLP-1 R assay (agonisticactivity, measured by amount of cAMP released).

FIG. 8 illustrates in vivo glucose reduction in C57 mice, with Seq. IDNo. 32, after intraperitonial (i.p) administration (dose response curve(DRC))

FIG. 9 illustrates in vivo glucose reduction in C57 mice, with Seq. IDNo. 32, after oral (p.o) administration (dose response curve (DRC))

FIG. 10 illustrates in vivo glucose reduction in db/db mice, with Seq.ID No. 32, after oral (p.o) administration.

FIG. 11 illustrates The serum insulin levels after single oraladministration of vehicles/test peptidomimetics (Seq. ID. No. 30, 31 and32), in C57BL/6J mice (in vivo).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, synthetic peptidomimeticshaving the structural formula (I), which showed glucose dependentinsulin secretion. Furthermore, it was found that these peptidomimeticsshowed glucagon receptor antagonistic activity, along with the GLP-1receptor agonistic activity. These dual acting peptidomimetics exhibitincreased stability to proteolytic cleavage, especially against DPP-IV(Dipeptidyl peptidase-IV) enzyme. Most of peptidomimetics were found tobe stable in rat plasma upto 24 hours (in vitro), showed increasedstability against GIT enzymes such as pepsin and acidic stomach pH andalso against liver microsomes (in vitro). Due to increased metabolicstability and also due to desirable net-charge profile, some of thesepeptidomimetics can also be delivered by oral routes of administration,for the treatment or prevention of diabetes and related metabolicdisorders.A-Z₁—Z₂—Z₃—Z₄—Z₅—Z₆—Z₇—Z₈—Z₉—Z₁₀—Z₁₁—B  (I)wherein, A represents the groups —NH—R₁, R₃—CO— or R₃—SO₂—, wherein R₁represents hydrogen, or optionally substituted linear or branched(C₁-C₁₀) alkyl chain; R₃ is selected from linear or branched (C₁-C₁₀)alkyl, (C₃-C₆) cycloallyl, aryl, heteroaryl or arylalkyl groups.

In a preferred embodiment, the aryl group is selected from phenyl,napthyl, indanyl, fluorenyl or biphenyl, groups; the heteroaryl group isselected from pyridyl, thienyl, furyl, imidazolyl, benzofuranyl groups;

B represents —COOR₂, —CONHR₂ or CH₂OR₂ or a tetrazole, wherein R₂represents H, optionally substituted groups selected from linear orbranched (C₁-C₁₀) alkyl group, aryl or aralkyl groups as definedearlier;

Z₁ represents Histidine (H);

Z₂ represents a naturally or unnaturally occurring amino acid selectedfrom the group comprising of L-Serine, D-Serine, L-alanine, D-alanine,α-amino-isobutyric acid (Aib), 1-amino cyclopropane carboxylic acid(ACP);

Z₃ represents glutamine (Gln; Q) or compounds of formula II.

Z₄ represents glycine (G) or the group 1-amino cyclopropane carboxylicacid (ACP);Z₅ represents a naturally or normaturally occurring amino acidcomprising a hydroxyl side chain; a preferred Z₅ is threonine orcompounds of formula III

Z₆ represents a naturally or unnaturally occurring amino acid having adisubstituted alpha carbon having two side chains, wherein each of themmay independently be an optionally substituted alkyl or aryl or anaralkyl group wherein the substituents may be selected from one or morealkyl groups or one or more halo groups. Preferred Z₆ representsphenylalanine (Phe; F), alpha-methyl-phenylalanine (-α-Me-Phe-),alpha-methyl-2-fluorophenylalanine (-α-Me-2F-Phe-) oralpha-methyl-2,6-difluorophenylalanine (-α-Me-2,6-F-Phe-) or2-fluorophenylalanine (-2F-Phe-) as given below.

Z₇ and Z₈ each independently represents a naturally or non-naturallyoccurring amino acid comprising a hydroxyl side chain, preferred Z₇ & Z₈is independently selected from threonine, serine, 1-amino cyclopropanecarboxylic acid (ACP) or compound of formula III as defined earlier;

Z₉ independently represent a naturally or normaturally occurring aminoacid having an amino acid side chain comprising an acidic group.Preferred Z₉ is selected from aspartic acid or compounds of formula IIas defined earlier.

Z₁₀ represents a naturally or unnaturally occurring amino acid offormula IV

Z₁₁ represents a naturally or unnaturally occurring amino acids offormula V (a-d)

DEFINITIONS

The term ‘natural amino acids’ indicates all those twenty amino acids,which are present in nature.

The term ‘unnatural amino acids’ or ‘non-natural amino acids’ representseither replacement of L-amino acids with corresponding D-amino acidssuch as replacement of L-Ala with D-Ala and the like or suitablemodifications of the L or D amino acids, amino alkyl acids, either by

-   -   α-alkylation such as substitution of Ala with α-methyl Ala (Aib)        or replacement of Phe with α-methyl Phe;    -   substitution on the side chain of amino acid such as        substitution of aromatic amino acid side chain with halogen,        (C₁-C₃)alkyl, aryl groups, more specifically the replacement of        Phe with 2 & 4-halo Phe;

The various groups, radicals and substituents used anywhere in thespecification are described in the following paragraphs.

The term “alkyl” used herein, either alone or in combination with otherradicals, denotes a linear or branched radical containing one to tencarbons, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, tert-butyl, amyl, t-amyl, n-pentyl, n-hexyl, iso-hexyl,heptyl, octyl, decyl and the like.

The term “cycloalkyl” used herein, either alone or in combination withother radicals, denotes a radical containing three to seven carbons,such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyland the like.

The term “aryl” or “aromatic” used herein, either alone or incombination with other radicals, denotes an aromatic system containingone, two or three rings wherein such rings may be attached together in apendant manner or may be fused, such as phenyl, naphthyl,tetrahydronaphthyl, indane, biphenyl, and the like.

The term “arylalkyl” denotes an alkyl group, as defined above, attachedto an aryl, such as benzyl, phenylethyl, naphthylmethyl, and the like.The term “aryloxy” denotes an aryl radical, as defined above, attachedto an alkoxy group, such as phenoxy, naphthyloxy and the like, which maybe substituted.

The term “aralkoxy” denotes an arylalkyl moiety, as defined above, suchas benzyloxy, phenethyloxy, naphthylmethyloxy, phenylpropyloxy, and thelike, which may be substituted.

The term “heteroaryl” or “heteroaromatic” used herein, either alone orin combination with other radicals, denotes an aromatic systemcontaining one, two or three rings wherein such rings may be attachedtogether in a pendant manner or may be fused containing one or morehetero atoms selected from O, N or S, such as pyridyl, thienyl, furyl,pyrrolyl, oxazolyl, thiazolyl, isothiazolyl, imidazolyl, isoxazolyl,oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzothienyl, indolinyl, indolyl,azaindolyl, azaindolinyl, benzodihydrofuranyl, benzodihydrothienyl,pyrazolopyrimidinyl, pyrazolopyrimidonyl, azaquinazolinyl,azaquinazolinoyl, pyridofuranyl, pyridothienyl, thienopyrimidyl,thienopyrimidonyl, quinolinyl, pyrimidinyl, pyrazolyl, quinazolinyl,quinazolonyl, pyrimidonyl, pyridazinyl, triazinyl, benzoxazinyl,benzoxazinonyl, benzothiazinyl, benzothiazinonyl, benzoxazolyl,benzothiazolyl, benzimidazolyl, benzotriazolyl, phthalazynil,naphthylidinyl, purinyl, carbazolyl, phenothiazinyl, phenoxazinyl, andthe like.

The term “heteroaralkyl” used herein, either alone or in combinationwith other radicals, denotes a heteroaryl group, as defined above,attached to a straight or branched saturated carbon chain containing 1to 6 carbons, such as (2-furyl)methyl, (3-furyl)methyl,(2-thienyl)methyl, (3-thienyl)methyl, (2-pyridyl)methyl,1-methyl-1-(2-pyrimidyl)ethyl and the like. The terms “heteroaryloxy”,“heteroaralkoxy”, “heterocycloxy denotes heteroaryl, heteroarylalkyl,groups respectively, as defined above, attached to an oxygen atom.

The term “acyl” used herein, either alone or in combination with otherradicals, denotes a radical containing one to eight carbons such asformyl, acetyl, propanoyl, butanoyl, iso-butanoyl, pentanoyl, hexanoyl,heptanoyl, benzoyl and the like, which may be substituted.

The term “carboxylic acid” used herein, alone or in combination withother radicals, denotes a —COOH group, and includes derivatives ofcarboxylic acid such as esters and amides. The term “ester” used herein,alone or in combination with other radicals, denotes —COO— group, andincludes carboxylic acid derivatives, where the ester moieties arealkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, and the like,which may be substituted.

Unless otherwise indicated, the term ‘amino acid’ as employed hereinalone or as part of another group includes, without limitation, an aminogroup and a carboxyl group linked to the same carbon, referred to as ‘α’carbon.

The absolute ‘S’ configuration at the ‘α’ carbon is commonly referred toas the ‘L’ or natural configuration. The ‘R’ configuration at the ‘α’carbon is commonly referred to as the ‘D’ amino acid. In the case whereboth the ‘α-substituents’ is equal, such as hydrogen or methyl, theamino acids are Gly or Aib and are not chiral.

The term ‘receptor antagonist’ refers to compounds that inhibit theactivation of receptor and generation of secondary messenger such ascyclic AMP either by competitive or non-competitive binding.

The term ‘Glucagon receptor antagonist’ refers to compounds that inhibitactivation of glucagon receptor.

The term ‘GLP-1 receptor modulator or agonist’ refers to a compound thatacts at the GLP-1 receptor to alter its ability to regulate downstreamsignaling events, such as cAMP production and insulin release. Exampleof receptor modulators includes agonist, partial agonist, inverseagonist and allosteric potentiators.

In accordance with the present invention, the synthetic isolatedpeptidomimetics described herein primarily act as a glucagon receptorantagonist. Furthermore, it was found that these peptidomimetics alsoact as GLP-1 receptor agonists. These synthetic peptidomimetics exhibitdesirable in vitro glucagon receptor antagonist properties as well asGLP-1 receptor agonist activity in CHO cells transfected with humanglucagon or GLP-1 receptor (H Glucagon R or HGLP-1R), in the range of1-100 nM concentration. H GLP-1 R agonistic activity, is assessed byestimation of amount of cAMP released, while glucagon antagonisticactivity was assessed by measuring the amount of cAMP productioninhibited by the test peptidomimetics, in presence of glucagon peptide.Novel peptidomimetics exhibit desirable in vitro glucagon receptorantagonist activity in CHO cells transfected with human glucagonreceptor, in the range of 1-100-nM concentration. Some of the testpeptidomimetics prepared showed glucose dependent insulin release andreduces fasting hyperglycemia, without causing hypoglycemia, when testedin vivo, in different diabetic animal models, such as hyperglycemic C57mice and db/db mice, thus making them ideal therapeutic candidates forthe treatment and prevention of type 2 diabetes. These new classes ofpeptidomimetics can be administered by oral or parenteral routes ofadministration.

The present invention provides peptidomimetics of formula (I)pharmaceutical compositions employing such peptidomimetics either aloneor in combination and for methods of using such peptidomimetics. Inparticular, the present invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of a peptidomimetics offormula (I), alone or in combination(s), with a pharmaceuticallyacceptable carrier.

Further provided is a method for treating or delaying the progression oronset of diabetes, especially type 2 diabetes, including complicationsof diabetes, including retinopathy, neuropathy, nephropathy and delayedwound healing and related diseases such as insulin resistance (impairedglucose homeostasis), hyperglycemia, hyperinsulinemia, elevated bloodlevels of fatty acids or glycerol, hyperlipidemia includinghypertriglyceridemia, syndrome X, atherosclerosis and hypertension,wherein a therapeutically effective amount of a peptidomimetics offormula (I) or their combination(s) are administered to a mammal,example, human, a patient in need of treatment.

Preparation of the Peptidomimetics:

Several synthetic routes can be employed to prepare the peptidomimeticsof the present invention well known to one skilled in the art of peptidesynthesis. The peptidomimetics of formula (I), where all symbols are asdefined earlier can be synthesized using the methods described below,together with conventional techniques known to those skilled in the artof peptide synthesis, or variations thereon as appreciated by thoseskilled in the art. Referred methods include, but not limited to thosedescribed below.

The peptidomimetics thereof described herein may be produced by chemicalsynthesis using suitable variations of various solid-phase techniquesgenerally known such as those described in G. Barany & R. B. Merrifield,“The peptides: Analysis, synthesis, Biology”; Volume 2-“Special methodsin peptide synthesis, Part A”, pp. 3-284, E. Gross & J. Meienhofer,Eds., Academic Press, New York, 1980; and in J. M. Stewart and J. D.Young, “Solid-phase peptide synthesis” 2nd Ed., Pierce chemical Co.,Rockford, Il, 1984.

The preferred strategy for preparing the peptidomimetics of thisinvention is based on the use of Fmoc-based SPPS approach, wherein Fmoc(9-Fluorenyl-methyl-methyloxycarbonyl) group is used for temporaryprotection of the α-amino group, in combination with the acid labileprotecting groups, such as t-butyloxy carbonyl (Boc), tert-butyl(Bu^(t)), Trityl (Trt) groups (FIG. 6), for temporary protection of theamino acid side chains (see for example E. Atherton & R. C. Sheppard,“The Fluorenylmethoxycarbonyl amino protecting group”, in “The peptides:Analysis, synthesis, Biology”; Volume 9—“Special methods in peptidesynthesis, Part C”, pp. 1-38, S. Undenfriend & J. Meienhofer, Eds.,Academic Press, San Diego, 1987).

The peptidomimetics can be synthesized in a stepwise manner on aninsoluble polymer support (resin), starting form the C-terminus of thepeptide. In an embodiment, the synthesis is initiated by appending theC-terminal amino acid of the peptide to the resin through formation ofan amide, ester or ether linkage. This allows the eventual release ofthe resulting peptide as a C-terminal amide, carboxylic acid or alcohol,respectively.

In the Fmoc-based SPPS, the C-terminal amino acid and all other aminoacids used in the synthesis are required to have their α-amino groupsand side chain functionalities (if present) differentially protected(orthogonal protection), such that the α-amino protecting group may beselectively removed during the synthesis, using suitable base such as20% piperidine solution, without any premature cleavage of peptide fromresin or deprotection of side chain protecting groups, usually protectedwith the acid labile protecting groups.

The coupling of an amino acid is performed by activation of its carboxylgroup as an active ester and reaction thereof with unblocked α-aminogroup of the N-terminal amino acid appended to the resin. After everycoupling and deprotection, peptidyl-resin was washed with the excess ofsolvents, such as DMF, DCM and diethyl ether. The sequence of α-aminogroup deprotection and coupling is repeated until the desired peptidesequence is assembled (Scheme 1). The peptide is then cleaved from theresin with concomitant deprotection of the side chain functionalities,using an appropriate cleavage mixture, usually in the presence ofappropriate scavengers to limit side reactions. The resulting peptide isfinally purified by reverse phase HPLC.

The synthesis of the peptidyl-resins required as precursors to the finalpeptides utilizes commercially available cross-linked polystyrenepolymer resins (Novabiochem, San Diego, Calif.). Preferred for use inthis invention are Fmoc-PAL-PEG-PS resin, 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetyl-p-methylbenzhydrylamine resin (Fmoc-Rink amide MBHA resin),2-chloro-Trityl-chloride resin or p-benzyloxybenzyl alcohol resin (HMPresin) to which the C-terminal amino acid may or may not be alreadyattached. If the C-terminal amino acid is not attached, its attachmentmay be achieved by HOBt active ester of the Fmoc-protected amino acidformed by its reaction with DIPCDI. In case of 2-Chloro-trityl resin,coupling of first Fmoc-protected amino acid was achieved, using DIPEA.For the assembly of next amino acid, N-terminal protection of peptidylresin was selectively deprotected using a solution of 10-20% piperidinesolution. After every coupling and deprotection, excess of amino acidsand coupling reagents were removed by washing with DMF, DCM and ether.Coupling of the subsequent amino acids can be accomplished using HOBt orHOAT active esters produced from DIPCDI/HOBt or DIPCDI/HOAT,respectively. In case of some difficult coupling, especially coupling ofthose amino acids, which are hydrophobic or amino acids with bulky sidechain protection, complete coupling can be achieved using a combinationof highly efficient coupling agents such as HBTU, PyBOP or TBTU, withadditives such as DIPEA.

The synthesis of the peptidomimetics described herein can be carried outby using batchwise or continuous flow peptide synthesis apparatus, suchas CS-Bio or AAPPTEC peptide synthesizer, utilizing the Fmoc/t-butylprotection strategy. The non-natural non-commercial amino acids presentat different position were incorporated into the peptide chain, usingone or more methods known in the art. In one approach, a Fmoc-protectednon-natural amino acid was prepared in solution, using appropriateliterature procedures. For example, the Fmoc-protected Bip analogs,described above, were prepared using modified Suzuki cross couplingmethod, as known in literature (Kotha, S., et al., Tetrahedron 2002, 58,9633). The Fmoc-protected α-methylated amino acids were prepared usingasymmetric Strecker synthesis (Boesten, W. H. J., et al., Org. Lett.,2001, 3(8), 1121). The resulting derivative was then used in thestep-wise synthesis of the peptide. Alternatively, the requirednon-natural amino acid was built on the resin directly using syntheticorganic chemistry procedures and a linear peptide chain were build.

The peptide-resin precursors for their respective peptidomimetics may becleaved and deprotected using suitable variations of any of the standardcleavage procedures described in the literature (King, D. S., et al.,Int. J. Peptide Protein Res., 1990, 36, 255). A preferred method for usein this invention is the use of TFA cleavage mixture, in the presence ofwater and TIPS as scavengers. Typically, the peptidyl-resin wasincubated in TFA/Water/TIPS (94:3:3; V:V:V; 10 ml/100 mg of peptidylresin) for 1.5-2 hrs at room temperature. The cleaved resin is thenfiltered off, the TFA solution is concentrated or dried under reducedpressure. The resulting crude peptide is either precipitated or washedwith Et₂O or is re-dissolved directly into DMF or 50% aqueous aceticacid for purification by preparative HPLC.

Peptidomimetics with the desired purity can be obtained by purificationusing preparative HPLC. The solution of crude peptide is injected into asemi-Prep column (Luna 10μ; C₁₈; 100 Å), dimension 250×50 mm and elutedwith a linear gradient of ACN in water, both buffered with 0.1% TFA,using a flow rate of 15-50 ml/min with effluent monitoring by PDAdetector at 220 nm. The structures of the purified peptidomimetics canbe confirmed by Electrospray Mass Spectroscopy (ES-MS) analysis.

All the peptide prepared were isolated as trifluoro-acetate salt, withTFA as a counter ion, after the Prep-HPLC purification. However, somepeptides were subjected for desalting, by passing through a suitable ionexchange resin bed, preferably through anion-exchange resin Dowex SBRP(Cl) or an equivalent basic anion-exchange resin. In some cases, TFAcounter ions were replaced with acetate ions, by passing throughsuitable ion-exchange resin, eluted with dilute acetic acid solution.For the preparation of the hydrochloride salt of peptides, in the laststage of the manufacturing, selected peptides, with the acetate salt wastreated with 4 M HCl. The resulting solution was filtered through amembrane filter (0.2 μm) and subsequently lyophilized to yield the whiteto off-white HCl salt. Following similar techniques and/or such suitablemodifications, which are well within the scope of persons skilled in theart, other suitable pharmaceutically acceptable salts of thepeptidomimetics of the present invention were prepared.

General Method of Preparation of Peptidomimetics, Using SPPS Approach:

Assembly of Peptidomimetics on Resin:

Sufficient quantity (50-100 mg) of Fmoc-PAL-PEG-PS resin or Fmoc-Rinkamide MBHA resin, loading: 0.5-0.6 mmol/g was swelled in DMF (1-10ml/100 mg of resin) for 2-10 minutes. The Fmoc-group on resin was thenremoved by incubation of resin with 10-30% piperidine in DMF (10-30ml/100 mg of resin), for 10-30 minutes. Deprotected resin was filteredand washed excess of DMF, DCM and ether (50 ml×4). Washed resin wasincubated in freshly distilled DMF (1 ml/100 mg of resin), undernitrogen atmosphere for 5 minutes. A 0.5 M solution of firstFmoc-protected amino acid (1-3 eq.), pre-activated with HOBt (1-3 eq.)and DIPCDI (1-2 eq.) in DMF was added to the resin, and the resin wasthen shaken for 1-3 hrs, under nitrogen atmosphere. Coupling completionwas monitored using a qualitative ninhydrin test. After the coupling offirst amino acid, the resin was washed with DMF, DCM and Diethyl ether(50 ml×4). For the coupling of next amino acid, firstly, theFmoc-protection on first amino acid, coupled with resin was deprotected,using a 10-20% piperidine solution, followed by the coupling theFmoc-protected second amino acid, using a suitable coupling agents, andas described above. The repeated cycles of deprotection, washing,coupling and washing were performed until the desired peptide chain wasassembled on resin, as per general Scheme 1 above.

Finally, the Fmoc-protected peptidyl-resin prepared above wasdeprotected by 20% piperidine treatment as described above and thepeptidyl-resins were washed with DMF, DCM and Diethyl ether (50 ml×4).Resin containing desired peptide was dried under nitrogen pressure for10-15 minutes and subjected for cleavage/deprotection.

Representative Example of Automated Solid Phase Synthesis of PeptideSequence ID. No. 32(H₂N-H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)-CONH₂).

The linear peptide chain,H₂N-H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)-PAL-PEG-PS wasassembled on an automated CS-Bio 536 PepSynthesiser™ using Fmoc solidphase peptide synthesis (SPPS) approach (Scheme 2). The Fmoc amino acidsand the2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate(TBTU) were packed together in vials and positioned in the amino acidmodule of the synthesizer. A stock solution of diisopropylethylamine(DIPEA; 0.9 M) and DMF were stored in reagent bottles, under drynitrogen atmosphere. The resin, Fmoc-PAL-PEG-PS (0.38 mmol/g; 1 g) wasdried over P₂O₅, in vacuo (1 hr) and swollen in freshly distilled DMF (5mL). The swollen resin was slurry packed into a glass column andpositioned in the synthesizer. All the synthetic cycles were carried outat a flow rate of 5 mL min⁻¹, Table 1. The resin was washed with freshlydistilled DMF for 10 minutes. Deprotection of Fmoc group was performedwith 20% piperidine in DMF for 10 minutes and the deprotection wasmonitored by UV detection of the column effluent at 304 nm.

Excess piperidine was removed by three auxiliary wash cycles and adistilled DMF wash cycle, with each cycle of 15 minutes. The amino groupwas treated with Fmoc-amino acid (4 equivalent), preactivated with TBTU(3.9 equivalent) in the presence of DIPEA (8 equivalent) and recycledfor 120 minutes. The excess amino acid and soluble by-products wereremoved from column and loop by four auxiliary wash cycles and distilledDMF wash cycles, with each cycle of 10 minutes. Furthermore, syntheticcycles (deprotection, wash, acylation and wash) were repeated forcomplete assembly of linear peptide. Final deprotection cycle wasperformed with 20% piperidine in DMF for 15 minutes to remove theterminal Fmoc group, followed by wash cycle (10×4 minutes). Completedpeptide-resin was filtered through sintered glass filter, washed threetimes successively with DMF, DCM, methanol, DMF and diethyl ether (100mL each). Peptide-resin was dried in vacuo over P₂O₅ (2 hr) and storedat −20° C. Ninhydrin resin test was carried out to check the N-terminalfree amino group of resin bound peptide. Appearance of blue-purplecolouration of the solution and the resin beads indicates the presenceof free amino group on resin bound peptide and was considered to be apositive test.

TABLE 1 Automated cycles for solid phase peptide synthesis Number ofTime Step Function Reagent/Solvent cycles (Minute) 1 WashDimethylformamide (DMF) 1 10 2 Deprotection 20% piperidine in DMF 2 15 3Wash DMF 3 15 4 Acylation Amino acid; TBTU and Recycle 120diisopropylethylamine (in DMF) 5 Wash Dimethylformamide 4 10

Small-scale cleavage was carried out to assess the purity of resin boundpeptide. The dried Peptide-resin (ca 10-mg) was treated with mixture(1-mL) of TFA, water, triisopropylsilane (95:2.5:2.5 v/v), for 90minutes at room temperature with gentle occasional swirling. The resinwas filtered, washed thoroughly with neat TFA (1 mL) and the entirefiltrate was evaporated under reduced pressure. Residual TFA wasazeotroped three times with diethyl ether (2 mL). Residue obtained wassuspended in distilled water (2 mL) and the aqueous layer was extractedthree times with diethyl ether (3 mL). The aqueous layer was separatedand freeze-dried to yield the crude peptideH₂N-H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)-CONH₂. Thelyophilised peptideH₂N-H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)-CONH₂ was dissolvedin 0.1% aqueous TFA (ca 1 mg/1 mL) and its purity was analyzed byanalytical RP-HPLC and characterized by electrospray ionisation massspectrometry (ESI-MS). Percent purity: 90% (crude peptide). ESI-MS;Calcd. for H₂N-H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)-CONH₂:1580 (M⁺), 1602 (M+Na⁺) and 1618 (M+K⁺); Found (m/z): 1580 (M⁺), 1602(M+Na⁺) and 1618 (M+K⁺).

Using above protocol and suitable variations thereof which are within,the scope of a person skilled in the art, the peptidomimetics designedin the present invention were prepared, using Fmoc-SPPS approach.Furthermore, resin bound peptidomimetics were cleaved and deprotected,purified and characterized using following protocol.

Cleavage and Deprotection:

The desired peptidomimetics were cleaved and deprotected from theirrespective peptidyl-resins by treatment with TFA cleavage mixture asfollows. A solution of TFA/Water/Triisopropylsilane (95:2.5:2.5) (10ml/100 mg of peptidyl-resin) was added to peptidyl-resins and themixture was kept at room temperature with occasional starring. The resinwas filtered, washed with a cleavage mixture and the combined filtratewas evaporated to dryness. Residue obtained was dissolved in 10 ml ofwater and the aqueous layer was extracted 3 times with ether (20 mleach) and finally the aqueous layer was freeze-dried. Crude peptideobtained after freeze-drying was purified by preparative HPLC asfollows:

Preparative HPLC Purification of the Crude Peptidomimetics:

Preparative HPLC was carried out on a Shimadzu LC-8A liquidchromatograph. A solution of crude peptide dissolved in DMF or water wasinjected into a semi-Prep column (Luna 10μ; C₁₈; 100 A⁰), dimension250×50 mm and eluted with a linear gradient of ACN in water, bothbuffered with 0.1% TFA, using a flow rate of 15-50 ml/min, with effluentmonitoring by PDA detector at 220 nm. A typical gradient of 20% to 70%of water-ACN mixture, buffered with 0.1% TFA was used, over a period of50 minutes, with 1% gradient change per minute. The desired producteluted were collected in a single 10-20 ml fraction and purepeptidomimetics were obtained as amorphous white powders bylyophilisation of respective HPLC fractions.

HPLC Analysis of the Purified Peptidomimetics

After purification by preparative HPLC as described above, each peptidewas analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLCsystem. For analytic HPLC analysis of peptidomimetics, Luna 5μ; C₁₈; 100Å°, dimension 250 X 4.6 mm column was used, with a linear gradient of0.1%. TFA and ACN buffer and the acquisition of chromatogram was carriedout at 220 nm, using a PDA detector.

Characterization by Mass Spectrometry

Each peptide was characterized by electrospray ionisation massspectrometry (ESI-MS), either in flow injection or LC/MS mode. Triplequadrupole mass spectrometers (API-3000 (MDS-SCIES, Canada) was used inall analyses in positive and negative ion electrospray mode. Full scandata was acquired over the mass range of quadrupole, operated at unitresolution. In all cases, the experimentally measured molecular weightwas within 0.5

Daltons of the calculated monoisotopic molecular weight. Quantificationof the mass chromatogram was done using Analyst 1.4.1 software.

Utilizing the synthetic methods described herein along with othercommonly known techniques and suitable variations thereof, the followingnovel peptidomimetics were prepared. This list is indicative of thevarious groups of peptidomimetics, which can be prepared according tothe present invention, and are expected to at least include obviousvariations of these peptidomimetics. However, such disclosure should notbe construed as limiting the scope of the invention in any way. In Table2-(i-vi), novel peptidomimetics of present invention are listed alongwith their corresponding Seq. ID. No.

TABLE 2 List of peptidomimetics prepared Seq. ID. No.Sequence of peptidomimetics (i): 5 HSQGTFTSD-Bip(OMe)-Bip(2Me) 6HSQGTFTSD-Bip(OMe)-Bip(Pyr) 7 HSQGTFTSD-Bip(OMe)-Bip(2F) 8HSQGTFTSD-Bip(OMe)-Bip(2CF₃) 9 HAQGTFTSD-Bip(OMe)-Bip(2Me) 10HAQGTFTSD-Bip(OMe)-Bip(Pyr) 11 HAQGTFTSD-Bip(OMe)-Bip(2F) 12HAQGTFTSD-Bip(OMe)-Bip(2CF₃) 13 H-Aib-QGTFTSD-Bip(OMe)-Bip(2Me) 14H-Aib-QGTFTSD-Bip(OMe)-Bip(Pyr) 15 H-Aib-QGTFTSD-Bip(OMe)-Bip(2F)  16H-Aib-QGTFTSD-Bip(OMe)-Bip(2CF₃) 17 H-(ACP)-QGTFTSD-Bip(OMe)-Bip(2Me) 18H-(ACP)-QGTFTSD-Bip(OMe)-Bip(Pyr) 19 H-(ACP)-QGTFTSD-Bip(OMe)-Bip(2F) 20H-(ACP)-QGTFTSD-Bip(OMe)-Bip(2CF₃) (ii) 21HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 22HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 23HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 24HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 25HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 26HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 27HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F)  28HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 29H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 30H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 31H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 32H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 33H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 34H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 35H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 36H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) (iii): 37HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 38HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 39HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 40HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 41HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 42HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(PYr) 43HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 44HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 45H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 46H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 47H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 48H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)  49H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 50H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 51H-(ACP)-QGT-(2F-Phe)-TSD7Bip(OMe)-Bip(2F) 52H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) (iv): 53HS-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me)  54 HS-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr)55 HS-(CNB)-GTFTSD-Bip(OMe)-Bip(2F) 56HS-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃) 57 HA-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me)58 HA-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr) 59 HA-(CNB)-GTFTSD-Bip(OMe)-Bip(2F)60 HA-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃) 61H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me) 62H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr) 63H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(2F) 64H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃) 65H-(ACP)-(CNB)-GTFTSD-Bip(OMe)43ip(2Me) 66H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr) 67H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(2F) 68H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃) 69HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 70HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 71HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 72HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 73HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 74HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 75HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 76HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 77H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 78H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 79H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 80H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 81H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 82H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 83H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 84H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)TSD-Bip(OMe)-Bip(2CF₃) 85HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 86HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 87HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 88HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 89HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 90HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 91HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 92HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 93H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 94H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 95H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 96H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 97H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 98H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 99H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-BiP(OMe)-Bip(2F) 100H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) (v): 101HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(2Me) 102 HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr)103 HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(2F) 104HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃) 105 HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(2Me)106 HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr) 107HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(2F) 108 HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃)109 H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2Me) 110H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr) 111H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2F) 112H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃) 113H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2Me) 114H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr) 115H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2F) 116H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃) 117HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 118HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 119HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 120HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 121HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 122HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 123HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 124HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 125H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 126H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 127H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 128H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 129H-(ACP)-Q-(ACP)-T (α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 130H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 131H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 132H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 133HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 134HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 135HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 136HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 137HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 138HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 139HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 140HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 141H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 142H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 143H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 144H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 145H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 146H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 147H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 148H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) (vi): 149HSQG-(PCA)-FTSD-Bip(OMe)-Bip(2Me) 150 HSQG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr)151 HSQG-(PCA)-FTSD-Bip(OMe)-Bip(2F) 152HSQG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃) 153 HAQG-(PCA)-FTSD-Bip(OMe)-Bip(2Me)154 HAQG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr) 155HAQG-(PCA)-FTSD-Bip(OMe)-Bip(2F) 156 HAQG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃)157 H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(2Me) 158H-Aib-QG-(PCA)-FTSD-Bip(OMet)-Bip(Pyr) 159H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(2F) 160H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃) 161H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(2Me) 162H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr) 163H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(2F) 164H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃) 165HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me)  166HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 167HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 168HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 169HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 170HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 171HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 172HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 173H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 174H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 175H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 176H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 177H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 178H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 179H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F) 180H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 181HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 182HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 183HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 184HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 185HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 186HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 187HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 188HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 189H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 190H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 191H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 192H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃) 193H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me) 194H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr) 195H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F) 196H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃)In Vitro and In Vivo Studies of Novel Peptidomimetics:

The peptidomimetics prepared as described above were tested for

-   a) In vitro glucose-dependent insulin secretion (RIN5F cell assay    screening protocol);-   b) In vitro Human GLP-1 R agonist activity (Cyclic AMP    determination);-   c) In vitro human glucagon antagonist activity (Cyclic AMP    determination);-   d) Stability of peptidomimetics against DPP IV enzyme, human plasma,    simulated gastric fluid, intestinal fluid and liver microsomes; and-   e) Demonstration of in vivo efficacy of test compounds    (peptidomimetics) in C57BL/6J mice (in vivo), using various in vitro    and in vivo assays, as described below.    In Vitro Studies:    In Vitro Glucose-Dependent Insulin Secretion (RIN5F Cell Assay    Screening Protocol)

RIN5F (Rat Insulinoma) cells were cultured in RPMI 1640 mediumsupplemented with sodium pyruvate (1 mM) HEPES and Glucose (4.5 g/L) ina humidified incubator (5% CO₂), at 37° C. After trypsinization, RIN5Fcells were seeded at a concentration of 0.2×10⁶·cells per well, in 12well plates. The cells were grown overnight to 80% confluence andinsulin secretion experiments were performed as follows(Montrose-Rafizadeh C., et al., Mol. Cell. Endo. 1997, 130, 109; Wang,X., et al., Endocrinology 2001, 5, 1820).

Cells were washed once with PBS solution followed by 40 min. incubationin fresh Krebs-Ringer Balanced Buffer containing NaCl (115 mmol/L), KCl(4.7 mmol/L), CaCl₂ (1.28 mmol/L), MgSO₄.7H₂O (1.2 mmol/L), KH₂PO₄ (1.2mmol/L), NaHCO₃ (10 mmol/L) and HEPES (25 mmol/L), containing Glucose(1.1 mM) and B.S.A (0.5%), pH 7.4. The buffer was replaced after 40 min.and the cells were incubated (37° C.) with the test peptidomimetics, atdifferent concentration, for 30 rain., both in the presence (16.7 mM)and absence (0 mM) of glucose load. The supernatant was collected andthe insulin amount was measured by ultra sensitive Rat insulin ELISA kit(Crystal Chem, Ill.). The protein was estimated in the supernatant usingBicinchoninic Acid kit, according to the manufacturer's protocol (SigmaAldrich, Mo.). The total insulin content obtained in Pico-gram (pg) wasdivided with the total protein (μg) in order to normalize fordifferences in cell density between wells. In vitro glucose dependentinsulin secretion activity of representative peptidomimetics are listedin [Table 3].

TABLE 3 In vitro glucose dependent insulin secretion activity ofrepresentative peptidomimetics Seq. ID. Conc. of test No. compd. (nM)Insulin secretion (pg/μg/hr)* Control 1 5.8 ± 0.66 (0 mM glucose)Control 2 10.6 ± 0.62 (16.7 mM glucose) Exendin-4 0.1/1/10 16.3 ±0.61/22.4 ± 0.52/36.5 ± 0.36 5 0.1/1/10 12.1 ± 0.52/14.1 ± 0.16/28.0 ±0.36 6 0.1/1/10 12.4 ± 0.26/15.2 ± 0.33/29.6 ± 0.51 7 0.1/1/10 11.1 ±0.12/13.1 ± 0.13/19.0 ± 0.16 8 0.1/1/10 12.6 ± 0.20/15.9 ± 0.31/28.4 ±0.11 9 0.1/1/10 11.8 ± 0.50/14.9 ± 0.11/27.9 ± 0.31 10 0.1/1/10 12.7 ±0.21/15.8 ± 0.33/29.3 ± 0.19 11 0.1/1/10 13.1 ± 0.11/14.9 ± 0.17/28.8 ±0.44 12 0.1/1/10 12.9 ± 0.14/15.8 ± 0.13/29.9 ± 0.15 13 0.1/1/10 16.1 ±0.22/22.1 ± 0.26/36.0 ± 0.36 14 0.1/1/10 15.4 ± 0.14/21.2 ± 0.18/35.6 ±0.17 15 0.1/1/10 15.6 ± 0.33/21.8 ± 0.16/35.6 ± 0.26 16 0.1/1/10 16.6 ±0.41/22.9 ± 0.32/36.7 ± 0.11 17 0.1/1/10 16.5 ± 0.12/22.7 ± 0.17/36.5 ±0.05 18 0.1/1/10 16.2 ± 0.13/22.4 ± 0.19/36.2 ± 0.09 19 0.1/1/10 17.1 ±0.15/23.1 ± 0.12/37.0 ± 0.19 20 0.1/1/10 16.9 ± 0.22/22.8 ± 0.31/36.7 ±0.34 21 0.1/1/10 12.3 ± 0.33/14.1 ± 0.36/28.0 ± 0.16 22 0.1/1/10 12.1 ±0.42/14.6 ± 0.41/27.8 ± 0.46 23 0.1/1/10 11.9 ± 0.17/14.2 ± 0.13/27.6 ±0.16 24 0.1/1/10 12.3 ± 0.33/14.8 ± 0.16/28.1 ± 0.22 25 0.1/1/10 12.4 ±0.22/15.2 ± 0.32/29.6 ± 0.50 26 0.1/1/10 12.1 ± 0.51/14.1 ± 0.19/28.1 ±0.29 27 0.1/1/10 12.6 ± 0.25/15.3 ± 0.31/29.7 ± 0.48 28 0.1/1/10 12.0 ±0.14/14.3 ± 0.12/27.8 ± 0.32 29 0.1/1/10 16.2 ± 0.20/22.2 ± 0.20/36.1 ±0.31 30 0.1/1/10 15.3 ± 0.19/21.2 ± 0.11/35.5 ± 0.19 31 0.1/1/10 15.6 ±0.31/21.8 ± 0.16/35.6 ± 0.28 32 0.1/1/10 16.6 ± 0.41/22.9 ± 0.32/36.6 ±0.19 33 0.1/1/10 16.4 ± 0.12/22.7 ± 0.17/36.6 ± 0.05 34 0.1/1/10 16.3 ±0.15/22.4 ± 0.21/36.2 ± 0.11 35 0.1/1/10 17.1 ± 0.11/23.1 ± 0.16/37.0 ±0.29 36 0.1/1/10 16.8 ± 0.20/22.7 ± 0.29/36.6 ± 0.31 *In vitro glucosedependent (16.7-mM glucose load) insulin secretion with variousconcentrations of peptidomimetics were measured using Rat Insulinoma(RIN5F) cells. The total insulin content (pg) was divided with totalprotein (μg) to normalize difference in cell density between wells. n =3, values represent mean ± .S.D. Basal insulin secretion was observedfor all the test compounds at 0-mM glucose concentration.In vitro Human GLP-1 R Agonist Activity (Cyclic AMP Determination).

The novel peptidomimetics were screened for Human GLP-1 receptor (HGLP-1R) agonist activity (in vitro), using the cAMP cell-based assay, instably transfected CHO/human GLP1R cells. The CHO-K1 cells (CRL 9618)were, obtained from American Type Culture Collection (Rockville, Md.).CHO cells were grown in Ham's F12 medium containing L-Glutamine (2 mM),HEPES (25 mM), NaHCO₃ (1.1 g/L) and supplemented with NewBorn Calf Serum(NBCS; 10%), Penicillin (50 U/ml (v/v)) and Streptomycin (50 ug/ml(v/v)). Cells were split every 3 days 1:8.

Production of Stable CHO Cell Lines Expressing the Human GLP-1 Receptor.

The cDNA encoding the human GLP-1 receptor was isolated by RT-PCRaccording to standard protocol. The full-length cDNA was cloned inpcDNA3.1(+). For the production of CHO cell lines expressing the GLP-1receptor, CHO cells were transfected with 10 μg of the expressionplasmid pcDNA/hGLP-1R using CaPO₄ according to the standard protocol(Wheeler, M. B., et al., Endocrinology 1993, 133, 57.). Clonesexpressing the receptor were generated by G418 (800 μg/ml active, Sigma)selection. The stable clones were thereafter maintained at 500 ug/ml(G418). The selected clone was used between passages 9-25 for cAMPassays.

Determination of cAMP Generation.

The CHO cells stably transfected with human GLP-1R were maintained inHam's F12+10% NBCS+500 ug/ml G418 upto a confluency of 70-75%. The cellswere trypsinized using 2 ml of TPVG (0.25% trypsin, 0:53 mM EDTA,1.38-mM glucose). The trypsin was inactivated using Ham's F12 mediumcontaining 10% NBCS and the cells were suspended in 2 ml of completemedium. 2×10⁵ cells/well were then seeded in 12 well plate and theplates were incubated in humidified atmosphere at 37° C. for 16-18 h(Fehmann, H. C., et al., Peptides 1994, 15, 453). The next day the assaywas proceeded, when the cells showed 90-95% confluency. The medium wasaspirated off from the 12 well plate and the cells were washed onceusing Ham's F12 (plain). The cells were incubated at 37° C. with 500 ulof Ham's F12+1% BSA+0.125 mM RO-20 for 30 min. After the incubation, themedium was aspirated off and fresh medium (plain Ham's F12+1% BSA+0.25mM RO-20) was added with 5 ul of test compounds (peptidomimetics) thathas been dissolved in water (MilliQ). The cells were incubated with thetest compounds for 30 min in humidified atmosphere and 37° C. After theincubation, the medium was removed and cells were washed once with plainHam's F12. Subsequently, the cells were lysed by adding 500 ul of icecold 0.1 N HCl to each well and shaking for 30 minutes at 200 rpm. Thecells were then scrapped, the lysate was collected in micro centrifugetubes and centrifuged at 12000 rpm for 10 min to remove the debris. 300ul of supernatant from each micro-centrifuge tube was then removed intoa glass tube and dried under N₂ for 30 min, for cAMP estimation. Thetotal cAMP was estimated from the sample according to the manufacturer'sprotocol using Cyclic AMP immunoassay kit (R&D systems, Minneapolis.MN). The remaining supernatant is used to determine the proteinconcentration using micro BCA (Sigma). Data is calculated as percent ofcontrol (Vehicle: water) and expressed as Mean±SD. The in-vitro humanGLP-1 receptor agonistic activities of representative peptidomimeticsare listed in Table 4.

TABLE 4 In vitro Human GLP-1 R activity (cAMP release) of test compounds(peptidomimetics), shown as % activity with respect to control. Seq. ID.Concentration of test compounds No. 1 nM 10 nM 100 nM 1 μM 10 μMExendin-4 89 ± 0.15 96 ± 0.18 99 ± 0.03 99 ± 0.09 99 ± 0.06 5 38 ± 0.1278 ± 0.15 86 ± 0.18 95 ± 0.03 98 ± 0.09 6 39 ± 0.11 80 ± 0.09 88 ± 0.0696 ± 0.14 99 ± 0.19 7  45 ± 0.022 84 ± 0.46 90 ± 0.41 99 ± 0.66 99 ±0.03 8 40 ± 0.09 81 ± 0.07 87 ± 0.04 95 ± 0.01 98 ± 0.08 9 38 ± 0.11 77± 0.16 85 ± 0.11 94 ± 0.08 97 ± 0.05 10 39 ± 0.10 81 ± 0.08 89 ± 0.09 96± 0.11 99 ± 0.16 11 51 ± 0.03 86 ± 0.40 91 ± 0.21 99 ± 0.32 99 ± 0.21 1255 ± 0.16 89 ± 0.05 93 ± 0.09 99 ± 0.02 99 ± 0.04 13 60 ± 0.12 92 ± 0.1598 ± 0.18 99 ± 0.03 99 ± 0.09 14 62 ± 0.11 94 ± 0.09 99 ± 0.06 99 ± 0.1499 ± 0.19 15  66 ± 0.022 97 ± 0.46 99 ± 0.41 99 ± 0.66 99 ± 0.03 16 69 ±0.09 98 ± 0.07 99 ± 0.04 99 ± 0.01 99 ± 0.08 17 78 ± 0.12 99 ± 0.15 99 ±0.18 99 ± 0.03 99 ± 0.09 18 86 ± 0.11 99 ± 0.09 99 ± 0.06 99 ± 0.14 99 ±0.19 19 96 ± 0.02 99 ± 0.46 99 ± 0.41 99 ± 0.66 99 ± 0.03 20 96 ± 0.0999 ± 0.07 99 ± 0.04 99 ± 0.01 99 ± 0.08 21 39 ± 0.12 80 ± 0.15 86 ± 0.1895 ± 0.03 98 ± 0.09 22 40 ± 0.11 81 ± 0.09 89 ± 0.06 96 ± 0.14 99 ± 0.1923  46 ± 0.022 85 ± 0.46 90 ± 0.41 99 ± 0.66 99 ± 0.03 24 41 ± 0.09 82 ±0.07 87 ± 0.04 95 ± 0.01 98 ± 0.08 25 38 ± 0.11 77 ± 0.16 85 ± 0.11 94 ±0.08 97 ± 0.05 26 39 ± 0.10 81 ± 0.08 89 ± 0.09 96 ± 0.11 99 ± 0.16 2782 ± 0.22 87 ± 0.12 92 ± 0.14 99 ± 0.22 99 ± 0.26 28 55 ± 0.16 88 ± 0.1392 ± 0.11 99 ± 0.07 99 ± 0.09 29 99 ± 0.12 99 ± 0.15 99 ± 0.18 99 ± 0.0399 ± 0.09 30 93 ± 0.11 99 ± 0.09 99 ± 0.06 99 ± 0.14 99 ± 0.19 31 93 ±0.10 99 ± 0.46 99 ± 0.41 99 ± 0.66 99 ± 0.03 32 99 ± 0.06 99 ± 0.06 99 ±0.08 99 ± 0.10 99 ± 0.12 33 99 ± 0.11 99 ± 0.13 99 ± 0.16 99 ± 0.06 99 ±0.10 34 99 ± 0.12 99 ± 0.08 99 ± 0.11 99 ± 0.12 99 ± 0.16 35 99 ± 0.0299 ± 0.26 99 ± 0.31 99 ± 0.60 99 ± 0.08 36 96 ± 0.09 99 ± 0.07 99 ± 0.0499 ± 0.01 99 ± 0.08

Based upon, the in-vitro human GLP-1 receptor agonistic activity, EC₅₀values were determined for novel peptidomimetics and the comparativedose-response curve (DRC) of Exendin and Seq. ID. No. 32 is shown inFIG. 7 as representative example.

In Vitro Human Glucagon Antagonist Activity (Measurement of Inhibitionof Amount of Cyclic AMP Production, with Test Peptidomimetics).

The novel peptidomimetics were screened for human glucagon receptor(H-glucagon-R) antagonistic activity (in vitro), using the cAMPcell-based assay, in stably transfected CHO/human glucagon R cells. TheCHO-K1 cells (CRL 9618) were obtained from American Type CultureCollection (Rockville, Md.). CHO cells were grown in Ham's F12 mediumcontaining L-Glutamine (2 mM), HEPES (25 mM), NaHCO₃ (1.1 g/L) andsupplemented with newborn Calf Serum (NBCS; 10%), Penicillin (50 U/ml(v/v)) and Streptomycin (50 ug/ml (v/v)). Cells were split every 3 days1:8.

Production of Stable CHO Cell Lines Expressing the Human GlucagonReceptor.

The cDNA encoding the human glucagon receptor was isolated by RT-PCRaccording to standard protocol. The full-length cDNA was cloned inpcDNA3.1(Invitrogen). For the production of CHO cell lines expressingthe glucagon receptor, CHO cells were transfected with 10 μg of theexpression plasmid pcDNA/H-glucagon-R using CaPO₄ according to thestandard protocol. Clones expressing the receptor were generated by G418(800 μg/ml active, Sigma) selection. The stable clones were thereaftermaintained at 500 ug/ml (G418). The selected clone was used betweenpassages 9-25 for cAMP assays.

Determination of Glucagon Antagonistic Activity by Measuring Amount ofcAMP Production Inhibited after Addition of Test Peptidomimetics Alongwith Glucagon Peptide.

The CHO cells stably transfected with human glucagon R were maintainedin Ham's F12+10% NBCS+500 ug/ml G418 upto a confluency of 70-75%. Thecells were trypsinized using 2 ml of TPVG (0.25% trypsin, 0.53 mM EDTA,1.38-mM glucose). The trypsin was inactivated using Ham's F12 mediumcontaining 10% NBCS and the cells were suspended in 2 ml of completemedium. 2×10⁵ cells/well were then seeded in 12 well plate and theplates were incubated in humidified atmosphere at 37° C. for 16-18 h.The next day the assay was proceeded, when the cells showed 90-95%confluency. The medium was aspirated off from the 12 well plate and thecells were washed once using Ham's F12 (plain). The cells were incubatedat 37° C. with 500 ul of Ham's F12+1% BSA+0.125 mM RO-20 for 30 min.After the incubation, the medium was aspirated off and fresh medium(plain Ham's F12+1% BSA+0.25 mM RO-20) was added with 5 ul of testcompounds (peptidomimetics) that has been dissolved in water (MilliQ),followed by addition of glucagon peptide (as agonist). The cells wereincubated with the peptidomimetics and glucagon peptide for 30 min inhumidified atmosphere and 37° C. After the incubation; the medium wasremoved and cells were washed once with plain Ham's F12. Subsequently,the cells were lysed by adding 500 ul of ice cold 0.1 N HCl to each welland shaking for 30 minutes at 200 rpm. The cells were then scrapped, thelysate was collected in micro centrifuge tubes and centrifuged at 12000rpm for 10 min to remove the debris. 300 ul of supernatant from eachmicro-centrifuge tube was then removed into a glass tube and dried underN₂ for 30 min, for cAMP estimation. The total cAMP was estimated fromthe sample according to the manufacturer's protocol using Cyclic AMPimmunoassay kit (R&D systems, Minneapolis: MN). The remainingsupernatant is used to determine the protein concentration using microBCA (Sigma). Data is calculated as percent of control (Vehicle: water)and expressed as Mean+SD. The in-vitro human glucagon receptorantagonistic activities of representative peptidomimetics are listed inTable 5.

TABLE 5 In vitro Human Glucagon receptor antagonistic activity of testcompounds (peptidomimetics) shown as inhibition of cAMP production(pmol/ml/μg prt) of Glucagon peptide, by the test compounds, incubatedat different concentration, along with saturated concentration ofglucagon peptide. Seq. ID. Concentration of test compounds No. 1 nM 10nM 100 nM 1 nM 10 μM Glucagon 22 ± 0.03  36 ± 0.07  36 ± 0.09  37 ±0.12  37 ± 0.08  5 20 ± 0.06  18 ± 0.09  16 ± 0.01  14 ± 0.03  12 ±0.01  6 22 ± 0.03  21 ± 0.05  20 ± 0.07  18 ± 0.04  18 ± 0.03  7 6 ±0.02 5 ± 0.03 2 ± 0.61 0 0 8 4 ± 0.01 2 ± 0.17 0 0 0 9 18 ± 0.07  16 ±0.01  12 ± 0.03  8 ± 0.02 6 ± 0.08 10 15 ± 0.11  12 ± 0.13  8 ± 0.09 4 ±0.01 2 ± 0.08 11 10 ± 0.03  8 ± 0.03 6 ± 0.22 2 ± 0.13 0 12 5 ± 0.09 3 ±0.11 0 0 0 13 10 ± 0.12  8 ± 0.02 6 ± 0.04 4 ± 0.05 1 ± 0.02 14 9 ± 0.116 ± 0.12 5 ± 0.14 3 ± 0.22 0 15 6 ± 0.01 5 ± 0.02 2 ± 0.13 0 0 16 4 ±0.02 2 ± 0.15 0 0 0 17 2 ± 0.03 0 0 0 0 18 0 0 0 0 0 19 5 ± 0.04 3 ±0.02 1 ± 0.11 0 0 20 5 ± 0.01 2 ± 0.17 0 0 0 21 19 ± 0.03  17 ± 0.08  15± 0.01  14 ± 0.02  12 ± 0.11  22 21 ± 0.11  19 ± 0.02  18 ± 0.07  18 ±0.03  16 ± 0.02  23 4 ± 0.05 2 ± 0.13 2 ± 0.12 0 0 24 4 ± 0.09 2 ± 0.120 0 0 25 18 ± 0.12  16 ± 0.11  12 ± 0.13  8 ± 0.12 6 ± 0.18 26 15 ±0.11  12 ± 0.13  8 ± 0.09 4 ± 0.01 2 ± 0.08 27 14 ± 0.02  11 ± 0.03  7 ±0.22 3 ± 0.12 0 28 4 ± 0.06 3 ± 0.11 0 0 0 29 10 ± 0.16  8 ± 0.12 6 ±0.14 4 ± 0.15 1 ± 0.12 30 9 ± 0.14 6 ± 0.14 5 ± 0.13 3 ± 0.24 0 31 6 ±0.05 5 ± 0.04 2 ± 0.16 0 0 32 4 ± 0.16 2 ± 0.11 0 0 0 33 0 0 0 0 0 34 00 0 0 0 35 5 ± 0.04 3 ± 0.15 1 ± 0.15 0 0 36 3 ± 0.12 1 ± 0.12 0 0 0Stability of Peptidomimetics Against DPP IV Enzyme, Human Plasma,Simulated Gastric Fluid, Intestinal Fluid and Liver Microsomes.

Different peptidomimetics (final concentration 2 μM) were incubated witheither DPP IV (1:25 mU) or pooled human plasma (7.5 μL) or simulatedgastric fluid (pH 1.5; composition HCl, NaCl and Pepsin) or simulatedintestinal fluid (pH 7.5) or human liver microsomes, for 0, 2, 4, 6, 12and 24 h (37° C.; 50 mM triethanolamine-HCl buffer; pH 7.8).Concentrations of DPP IV enzyme/human plasma/simulated gastricfluid/simulated intestinal fluid/human liver microsomes were selected inpreliminary experiments to provide degradation of approximately 50% ofExendin within 2-4 h, therefore allowing time-dependent degradation tobe viewed over 24 h. Reactions were terminated by the addition ofTFA/H₂O (15 mL, 10% (v/v)). The reaction products were then applied to aVydac C₁₈ analytical column (4.6×250-mm) and the major degradationfragment separated from intact peptidomimetic. The column wasequilibrated with TFA/H₂O, at a flow rate of 1 mL/min. Using 0.1% (v/v)TFA in 70% acetonitrile/H₂O, the concentration of acetonitrile in theeluting solvent was raised from 0% to 28% over 10 min and from 28% to42% over 30 min. The absorbance was monitored at 206 nm using UVdetector and peaks were collected manually prior to ESI-MS analysis.Area under the curve was measured for test peptidomimetics and theirmetabolites and percentage degradation were calculated at each timepoint over a period of 24 h. Stability study results of selectedpeptidomimetics, against DPP IV enzyme, human plasma, simulated gastricfluid, intestinal fluid and liver microsomes (in vitro) are listed inTable 6.

TABLE 6 Stability study results of selected peptidomimetics against DPPIV enzyme, human plasma, simulated gastric fluid, intestinal fluid andliver microsomes (in vitro) Seq. Simulated Simulated ID. DPP IV Humangastric intestinal liver No. enzyme^(a) plasma^(b) fluid^(c) fluid^(d)microsomes^(e) EX-4 89 (6) 86 (6.2) 100 (0.3) 100 (0.3) 100 (0.4) 5 76(10) 78 (9) 100 (0.5) 100 (0.5) 100 (0.5) 6 75 (10) 77 (9) 100 (0.5) 100(0.5) 100 (0.5) 7 77 (10) 80 (9) 100 (0.5) 100 (0.5) 100 (0.5) 8 76 (10)78 (9) 100 (0.5) 100 (0.5) 100 (0.5) 9 74 (10) 75 (9) 100 (0.5) 100(0.5) 100 (0.5) 10 70 (10) 71 (9) 100 (0.5) 100 (0.5) 100 (0.5) 11 86(10) 70 (9) 100 (0.5) 100 (0.5) 100 (0.5) 12 72 (10) 70 (9) 100 (0.5)100 (0.5) 100 (0.5) 13 00 (>24) 00 (>24) 50 (4) 00 (>24) 86 (2) 14 00(>24) 00 (>24) 55 (4) 00 (>24) 84 (2) 15 00 (>24) 00 (>24) 45 (4) 00(>24) 85 (2) 16 00 (>24) 00 (>24) 43 (4) 00 (>24) 84 (2) 17 00 (>24) 00(>24) 49 (4) 00 (>24) 82 (2) 18 00 (>24) 00 (>24) 52 (4) 00 (>24) 81 (2)19 00 (>24) 00 (>24) 43 (4) 00 (>24) 84 (2) 20 00 (>24) 00 (>24) 41 (4)00 (>24) 80 (2) 21 76 (9) 78 (8) 12 (8) 55 (6) 79 (1) 22 75 (9) 77 (8)14 (8) 45 (6) 81 (1) 23 77 (9) 80 (8) 13 (8) 50 (6) 82 (1) 24 76 (9) 78(8) 14 (8) 43 (6) 80 (1) 25 74 (9) 75 (8) 12 (8) 46 (6) 83 (1) 26 70 (9)71 (8) 14 (8) 40 (6) 78 (1) 27 86 (9) 70 (8) 15 (8) 41 (6) 77 (1) 28 72(9) 70 (8) 12 (8) 42 (6) 78 (1) 29 00 (>24) 00 (>24) 00 (>24) 00 (>24)35 (5) 30 00 (>24) 00 (>24) 00 (>24) 00 (>24) 33 (5) 31 00 (>24) 00(>24) 00 (>24) 00 (>24) 31 (5) 32 00 (>24) 00 (>24) 00 (>24) 00 (>24) 32(5) 33 00 (>24) 00 (>24) 00 (>24) 00 (>24) 33 (5) 34 00 (>24) 00 (>24)00 (>24) 00 (>24) 32 (5) 35 00 (>24) 00 (>24) 00 (>24) 00 (>24) 26 (5)36 00 (>24) 00 (>24) 00 (>24) 00 (>24) 35 (5) ^(a)% degradation ofpeptidomimetics in 24 h when incubated with DPP-IV enzyme and values inbracket represent half-life (t_(1/2)), in h; ^(b)% degradation ofpeptidomimetics in 24 h when incubated with human plasma and values inbracket represent half-life (t_(1/2)), in h; ^(c)% degradation ofpeptidomimetics in 24 h when incubated with simulated gastric fluid andvalues in bracket represent half-life (t_(1/2)), in h; ^(d)% degradationof peptidomimetics in 24 h when incubated with simulated intestinalfluid and values in bracket represent half-life (t_(1/2)), in h; ^(e)%degradation of peptidomimetics in 24 h when incubated with livermicrosomes and values in bracket represent half-life (t_(1/2)), in h.In Vivo Efficacy Studies:Demonstration of In Vivo Efficacy (Antihyperglycaemic/AntidiabeticActivity) of Test Compounds (Peptidomimetics) in CS7BL/6J or db/db Mice,Both by Parenteral (i.p) and Oral Routes of Administration.Animals

Acute single dose 120-min time-course experiments were carried out inmale C57BL/6J or db/db mice, age 8-12 weeks, bred in-house. Animals werehoused in groups of 6 animals per cage, for a week, in order tohabituate them to vivarium conditions (25±4° C., 60-65% relativehumidity, 12:12 h light:dark cycle, with lights on at 7.30 am). All theanimal experiments were carried out according to the internationallyvalid guidelines following approval by the ‘Zydus Research Center animalethical committee’.

Procedure

The in-vivo glucose lowering properties of some of the test compounds(peptidomimetics) and Exendin-4 were evaluated in C57BL/6J (mildhyperglycemic) or db/db animal models as described below. Two days priorto the study, the animals were randomised and divided into 5 groups(n=6), based upon their fed glucose levels. On the day of experiment,food was withdrawn from all the cages, water was given ad-libitum andwere kept for overnight fasting. Vehicle (normal saline)/test/standardcompounds were administered intraperitoneally (i.p.) or orally, on abody weight basis. Soon after the 0 min. blood collection from eachanimal, the subsequent blood collections were done at 30, 60 and 120 orupto 240 min., via retro-orbital route, under light ether anesthesia(Chen, D., et al., Diabetes Obesity Metabolism, 2005, 7, 307. Kim, J. G.et al., Diabetes, 2003, 52, 751).

Blood samples were centrifuged and the separated serum was immediatelysubjected for the glucose estimation. Serum for insulin estimation wasstored at −70° C. until used for the insulin estimation. The glucoseestimation was carried out with DPEC-GOD/POD method (Ranbaxy FineChemicals Limited, Diagnostic division, India), using Spectramax-190, in96-microwell plate reader (Molecular devices Corporation, Sunnyvale,Calif.). Mean values of duplicate samples were calculated usingMicrosoft excel and the Graph Pad Prism software (Ver. 4.0) was used toplot a 0 min base line corrected line graph, area under the curve (0-120min AUC) and base line corrected area under the curve (0 min BCAUC). TheAUC and BCAUC obtained from graphs were analyzed for one way ANOVA,followed by Dunnett's post test, using Graph Pad prism software.Furthermore, the insulin estimation was carried out using rat/mouseinsulin ELISA kit (Linco research, Mo. USA). Changes in the bloodglucose levels, at 0, 30, 60 and 120 min, with selected peptidomimeticsare shown in Table 7 (via ip route of administration) and Table 8 (viaoral route of administration) respectively.

TABLE 7 Acute single dose 120-min time-course experiments, in maleC57BL/6J mice (in vivo glucose reduction); n = 8, all values are Mean ±SEM, via intraperitonial (i.p.) route of administration. Treatment group0 min 30 min 60 min 120 min C57 control 183 ± 6.2 186 ± 6.1 188 ± 5.2181 ± 5.4 Exendin 182 ± 5.3 111 ± 4.2 128 ± 2.1 145 ± 1.2 (2 nM/kg, i.p)Seq. ID. 5 181 ± 5.1 120 ± 3.8 128 ± 2.2 149 ± 1.6 (50 nM/kg, i.p) Seq.ID. 6 180 ± 5.2 119 ± 3.2 129 ± 2.6 148 ± 1.8 (50 nM/kg, i.p) Seq. ID. 7182 ± 5.3 121 ± 3.3 127 ± 2.8 149 ± 2.0 (50 nM/kg, i.p) Seq. ID. 8 183 ±5.1 122 ± 3.1 130 ± 2.3 148 ± 2.2 (50 nM/kg, i.p) Seq. ID. 9 180 ± 5.2120 ± 2.9 138 ± 2.4 149 ± 2.0 (50 nM/kg, i.p) Seq. ID. 10 181 ± 5.3 119± 3.0 128 ± 2.6 147 ± 1.9 (50 nM/kg, i.p) Seq. ID. 11 179 ± 5.0 120 ±3.6 129 ± 2.0 148 ± 1.7 (50 nM/kg, i.p) Seq. ID. 12 184 ± 5.0 122 ± 3.8128 ± 2.1 149 ± 2.3 (50 nM/kg, i.p) Seq. ID. 13 182 ± 5.1 118 ± 2.1 116± 2.2 118 ± 2.4 (30 nM/kg, i.p) Seq. ID. 14 181 ± 5.2 119 ± 2.6 117 ±2.1 119 ± 2.3 (30 nM/kg, i.p) Seq. ID. 15 180 ± 5.3 118 ± 2.8 118 ± 2.4118 ± 2.1 (30 nM/kg, i.p) Seq. ID. 16 183 ± 4.9 117 ± 2.5 116 ± 2.3 119± 2.0 (30 nM/kg, i.p) Seq. ID. 17 182 ± 4.8 118 ± 2.8 116 ± 2.6 118 ±1.2 (30 nM/kg, i.p) Seq. ID. 18 181 ± 4.1 117 ± 3.8 117 ± 3.0 117 ± 1.6(30 nM/kg, i.p) Seq. ID. 19 180 ± 3.2 119 ± 3.2 117 ± 3.1 118 ± 1.8 (30nM/kg, i.p) Seq. ID. 20 183 ± 3.6 118 ± 3.3 116 ± 3.2 117 ± 1.9 (30nM/kg, i.p) Seq. ID. 21 182 ± 3.3 120 ± 3.6 127 ± 3.4 145 ± 2.1 (20nM/kg, i.p) Seq. ID. 22 181 ± 3.0 121 ± 3.4 128 ± 2.8 141 ± 2.3 (20nM/kg, i.p) Seq. ID. 23 180 ± 3.1 122 ± 3.9 129 ± 2.2 140 ± 2.9 (20nM/kg, i.p) Seq. ID. 24 179 ± 2.9 121 ± 3.0 129 ± 3.8 142 ± 1.6 (20nM/kg, i.p) Seq. ID. 25 182 ± 2.8 122 ± 3.2 128 ± 3.4 141 ± 1.7 (20nM/kg, i.p) Seq. ID. 26 181 ± 3.0 123 ± 3.8 128 ± 3.2 146 ± 1.8 (20nM/kg, i.p) Seq. ID. 27 183 ± 4.9 120 ± 2.8 129 ± 3.0 139 ± 2.0 (20nM/kg, i.p) Seq. ID. 28 182 ± 2.1 121 ± 2.9 130 ± 3.1 138 ± 2.4 (20nM/kg, i.p) Seq. ID. 29 180 ± 2.5 110 ± 2.6 111 ± 3.2 112 ± 2.3 (10nM/kg, i.p) Seq. ID. 30 182 ± 2.1 111 ± 2.1 112 ± 2.8 113 ± 2.2 (10nM/kg, i.p) Seq. ID. 31 183 ± 2.2 113 ± 2.8 113 ± 2.9 114 ± 2.0 (10nM/kg, i.p) Seq. ID. 32 182 ± 2.3 110 ± 2.3 112 ± 3.0 112 ± 1.9 (10nM/kg, i.p) Seq. ID. 33 181 ± 2.5 111 ± 3.4 113 ± 3.1 110 ± 1.8 (10nM/kg, i.p) Seq. ID. 34 180 ± 4.2 113 ± 3.2 112 ± 2.4 110 ± 1.9 (10nM/kg, i.p) Seq. ID. 35 182 ± 4.1 112 ± 3.1 113 ± 2.6 111 ± 1.8 (10nM/kg, i.p) Seq. ID. 36 180 ± 2.8 110 ± 3.6 112 ± 2.3 112 ± 1.6 (10nM/kg, i.p)

TABLE 8 Acute single dose 120-min time-course experiments, in maleC57BL/6J mice (in vivo glucose reduction), with selectedpeptidomimetics; n = 8, all values are Mean ± SEM, via oral route ofadministration Treatment group 0 min 30 min 60 min 120 min C57 control183 ± 2.2 186 ± 3.1 189 ± 4.2 182 ± 2.4 Seq. ID. 29 184 ± 2.5 115 ± 3.6113 ± 4.2 110 ± 2.4 (2 μM/kg, oral) Seq. ID. 30 183 ± 2.4 116 ± 3.1 112± 4.8 110 ± 2.5 (2 μM/kg, oral) Seq. ID. 31 185 ± 2.3 115 ± 3.8 113 ±4.9 109 ± 2.2 (2 μM/kg, oral) Seq. ID. 32 181 ± 2.5 117 ± 3.3 112 ± 4.0108 ± 2.9 (2 μM/kg, oral) Seq. ID. 33 182 ± 2.5 116 ± 3.1 113 ± 4.1 110± 2.8 (2 μM/kg, oral) Seq. ID. 34 184 ± 2.2 118 ± 3.3 112 ± 4.4 110 ±2.9 (2 μM/kg, oral) Seq. ID. 35 183 ± 2.1 118 ± 3.4 113 ± 4.6 111 ± 2.8(2 μM/kg, oral) Seq. ID. 36 183 ± 2.3 119 ± 3.0 112 ± 4.3 109 ± 2.6 (2μM/kg, oral)

Some of the baseline corrected serum glucose levels, as representativefigures are shown, after single dose treatment with Seq. ID. No. 32, atdifferent doses (DRC), either in C57, via ip (FIG. 8), oral (FIG. 9) orin db/db (FIG. 10) via oral route of administration, while FIG. 11represent the change in serum insulin levels after single oraladministration of vehicles/test compounds (Seq. ID. No. 30, 31 and 32),in C57BL/6J mice (in vivo).

Overview on In Vitro and In Vivo Results of Peptidomimetics:

As described above, all the peptidomimetics prepared in the presentinvention were evaluated in vitro and in vivo and the data of selectedpeptidomimetics were presented in above section as examples ofrepresentative peptidomimetics. In RIN 5F (rat insulinoma) cell basedassay, all the peptidomimetics showed only glucose-dependent insulinsecretion, in the range of 1-10 nM concentration (Table 3), therebythese class of peptidomimetics are likely to be devoid of hyperglycemicepisodes, which is commonly observed with other class of insulinsecretagogues, such as sulfonylureas. In human glucagon receptor assay,in vitro antagonistic activity of peptidomemtics were estimated bymeasuring the inhibition of amount of cAMP production, with testpeptidomemtics, when incubated along with the glucagon peptide. As shownin the Table 5, in general, all the peptidomimetics showed significantglucagon receptor antagonistic activity, in the range of 1 nM to 1000nM. In HGLP-1R assay, the novel peptidomimetics showed concentrationdependent cAMP production (in vitro GLP-1 agonist activity), in therange of 1-100 nM concentration (Table 4). This dual nature ofpeptidomimetics (antagonist of the glucagon receptor and agonist of theGLP-1 receptor), make them ideal candidate for the safe and effectivetreatment of type 2 diabetes and associated metabolic disorders.

Stability study results of selected peptidomimetics against DPP-IVenzyme, human plasma, simulated gastric and intestinal fluid and livermicrosomes, indicates that most of the peptidomimetics are stableagainst DPP-IV enzyme, when incubated upto 24 hrs. Similarly, in humanplasma, simulated gastric and intestinal fluid, most of thepeptidomimetics were found to be stable, when incubated upto 24 hrs.Incubation of peptidomimetics with liver microsomes showed significantstability and only 26-35% degradation were observed in 24 hrs, indicatedthat some of the peptidomimetics could be delivered by oral route ofadministration.

In vivo antihyperglycaemic/antidiabetic activity of peptidomimetics,both by parenteral and oral route of administration were determined inC57 or db/db mice, using acute-single-dose 120/240-min time courseexperiment. As shown in Table 7, most of the peptidomimetics are activevia i.p. route of administration, in the dose range of 10-50 nM, whileorally, some of the selected peptidomimetics (Table 8) are active in therange of 1-2 μM/kg dose. Thus novel peptidomimetics exhibit glucagonantagonistic and GLP-1 agonistic activity and are orally bioavailable,which make them ideal candidate for the safe and effective treatment oftype 2 diabetes and associated metabolic disorders.

Utilities:

In a preferred embodiment, the present invention provides a method ofmaking a peptidomimetic, that function both as an antagonist of theglucagon receptor and agonist of the GLP-1 receptor having differentdegree of affinity/selectivity towards both the receptors and useful forreducing circulating glucose levels & for the treatment of diabetes.

The synthetic peptidomimetics described in the present embodimentexhibit desirable in vitro Glucagon antagonistic and GLP-1 agonistactivity in CHO cells transfected with human glucagon or HGLP-1R, in nMconcentration, and in vivo, some of the peptidomimetics showed glucosedependent insulin release and reduces fasting hyperglycemia, withoutcausing hypoglycemia, when tested in different diabetic animal models,such as hyperglycemic C57 mice and db/db mice.

Novel peptidomemtics of present invention showed increased stabilityagainst various proteolytic enzymes and due to increased stability andshort chain length, such peptidomimetics can also be delivered by oralroute of administration, along with other invensive and non-invensiveroutes of administration.

The novel peptidomimetics of the present invention can be formulatedinto suitable pharmaceutically acceptable compositions by combining withsuitable excipients as are well known.

The pharmaceutical composition is provided by employing conventionaltechniques. Preferably the composition is in unit dosage form containingan effective amount of the active component, that is, thepeptidomimetics of formula (I) either alone or combination, according tothis invention.

The quantity of active component, that is, the peptidomimetics offormula (I) according to this invention, in the pharmaceuticalcomposition and unit dosage form thereof may be varied or adjustedwidely depending upon the particular application method, the potency ofthe particular peptidomimetics and the desired concentration. Generally,the quantity of active component will range between 0.5% to 90% byweight of the composition.

Accordingly, the peptidomimetics of the present invention can beadministered to mammals, preferably humans, for the treatment of avariety of conditions and disorders, including, but not limited to,treating or delaying the progression or onset of diabetes (preferablytype II, impaired glucose tolerance, insulin resistance and diabeticcomplications, such as nephropathy, retinopathy, neuropathy andcataracts), hyperglycemia, hyperinsulinemia, hypercholesterolemia,elevated blood levels of free fatty acids or glycerol, hyperlipidemia,hypertriglyceridemia, wound healing, tissue ischemia, atherosclerosis,hypertension, intestinal diseases (such as necrotizing enteritis,microvillus inclusion disease or celic disease). The peptidomimetics ofthe present invention may also be utilized to increase the blood levelsof high-density lipoprotein (HDL).

In addition, the conditions, diseases collectively referenced to as‘Syndrome X’ or metabolic syndrome as detailed in Johannsson G., J.,Clin. Endocrinol. Metab., 1997, 82, 727, may be treated employing thepeptidomimetics of the invention. The peptidomimetics of the presentinvention may optionally be used in combination with suitable DPP-IVinhibitors for the treatment of some of the above disease states eitherby administering the compounds sequentially or as a formulationcontaining the peptidomimetics of the present invention along with asuitable DPP-IV inhibitors.

No adverse effects were observed for any of the mentionedpeptidomimetics of invention. The compounds of the present inventionshowed good glucose serum-lowering activity in the experimental animalsused. These peptidomimetics are used for the testing/prophylaxis ofdiseases caused by hyperinsulinemia, hyperglycemia such as NIDDM,metabolic disorders, since such diseases are inter-linked to each other.

We claim:
 1. An isolated peptidomimetic having a sequence of Formula(I), or a tautomer or solvate thereof;A-Z₁—Z₂—Z₃—Z₄—Z₅—Z₆—Z₇—Z₈—Z₉—Z₁₀—Z₁₁—B  (I) wherein, A represents thegroups —NH—R₁, R₃—CO— or R₃—SO₂—, wherein R₁ represents hydrogen, oroptionally substituted linear or branched (C₁-C₁₀) alkyl chain; R₃ isselected from linear or branched (C₁-C₁₀) alkyl, (C₃-C₆) cycloalkyl,aryl, heteroaryl or arylalkyl groups; B represents the groups —COOR₂,—CONHR₂ or CH₂OR₂ or a tetrazole, wherein R₂ represents H, optionallysubstituted groups selected from linear or branched (C₁-C₁₀) alkyl, arylor aralkyl groups; Z₁ represents Histidine; Z₂ represents a naturally orunnaturally occurring amino acid selected from the group comprisingL-Serine, D-Serine, L-alanine, D-alanine, α-amino-isobutyric acid, and1-amino cyclopropane carboxyl acid; Z₃ represents glutamine (Gln; Q) orcompounds of formula II;

Z₄ represents glycine or the group 1-amino cyclopropane carboxylic acid;Z₃ represents a naturally or non-naturally occurring amino acidcomprising a hydroxyl side chain; Z₆ represents, a naturally ofunnaturally occurring amino acid having a disubstituted alpha carbonhaving two side chains, wherein each of them are independently selectedfrom an optionally substituted alkyl, aryl or an aralkyl group whereinthe substituents are selected from one or more alkyl groups or one ormore halo groups; Z₇ and Z₈ independently represents a naturally ornon-naturally occurring amino acid comprising a hydroxyl side chain; Z₉independently represents a naturally or non-naturally occurring aminoacid having an amino acid side chain comprising an acidic group; Z₁₀represents a naturally or unnaturally occurring amino acid of formula IV

and Z₁₁ represents a naturally or unnaturally occurring amino acid offormula V(a-d)


2. The compound of formula I as claimed in claim 1, wherein Z₅ isthreonine.
 3. The compound of formula I as claimed in claim 1, whereinZ₅ represents a compound of formula III


4. The compound of formula I as claimed in claim 1, wherein Z₆represents Phe (F), alpha-methyl-phenylalanine (-α-Me-Phe-),alpha-methyl-2-fluorophenylalanine (-α-Me-2F-Phe-),alpha-methyl-2,6-diflurophenylalanine (-α-Me-2,6-F-Phe-) or2-fluorophenylalanine (-2F-Phe-).
 5. The compound of formula I asclaimed in claim 1 wherein each of Z₇ and Z₈ is independently selectedfrom threonin, serine, 1-amino cyclopropane carboxylic acid or acompound of formula III


6. The compound of formula I as claimed in claim 1, wherein Z₉ isselected from aspartic acid or a compound of formula II


7. The compound of formula IA-Z₁—Z₂—Z₃—Z₄—Z₅—Z₆—Z₇—Z₈—Z₉—Z₁₀—Z₁₁—B  (I) wherein A represents thegroups —NH—R₁, R₃—CO— or R₃—SO₂—, wherein R₁ represents hydrogen, oroptionally substituted linear or branched (C₁-C₁₀) alkyl chain, R₃ isselected from linear or branched (C₁-C₁₀) alkyl, (C₃-C₆) cycloalkyl,aryl, heteroaryl or arylalkyl groups; B represents —COOR₂, —CONHR₂ orCH₂OR₂ or a tetrazole, wherein R₂ represents H, optionally substitutedgroups selected from linear or branched (C₁-C₁₀) alkyl group, aryl oraralkyl groups; Z₁ represents Histidine (H); Z₂ is selected fromL-Serine, D-Serine, L-alanine, D-alanine, α-amino-isobutyric acid,I-amino cyclopropane carboxylic acid; Z₃ represents glutamine (Gln; Q)or a compound of formula II

Z₄ represents glycine or the group 1-amino cyclopropane carboxylic acid;Z₅ is selected from threonine or compounds of formula III

Z₆ is selected from Phe (F), alpha-methyl-phenylalanine (-α-Me-Phe-),alpha-methyl-2-fluorophenylalanine (-α-Me-2F-Phe-),alpha-methyl-2,6-diflurophenylalanine (-α-Me-2,6-F-Phe-) or2-fluorophenylalanine (-2F-Phe-); Z₇ and Z₈ each is independentlyselected from threonine, serine, 1-amino cyclopropane carboxylic acid orcompound of formula III as defined earlier; Z₉ is selected from Asparticacid or compounds of formula II as defined earlier; Z₁₀ represents anaturally or unnaturally occurring amino acid of formula IV

and Z₁₁ selected from amino acids of formula V (a-d)


8. The compound of formula I as claimed in claim 1, wherein the arylgroup is selected from phenyl, napthyl, indanyl, fluorenyl and biphenylgroups.
 9. The compound of formula I as claimed in claim 1, wherein theheteroaryl group is selected from pyridyl, thienyl, furyl, imidazolyl,and benzofuranyl groups.
 10. A compound selected from:HSQGTFTSD-Bip(OMe)-Bip(2Me); HSQGTFTSD-Bip(OMe)-Bip(Pyr);HSQGTFTSD-Bip(OMe)-Bip(2F); HSQGTFTSD-Bip(OMe)-Bip(2CF₃);HAQGTFTSD-Bip(OMe)-Bip(2Me); HAQGTFTSD-Bip(OMe)-Bip(Pyr);HAQGTFTSD-Bip(OMe)-Bip(2F); HAQGTFTSD-Bip(OMe)-Bip(2CF₃);H-Aib-QGTFTSD-Bip(OMe)-Bip(2Me); H-Aib-QGTFTSD-Bip(OMe)-Bip(Pyr);H-Aib-QGTFTSD-Bip(OMe)-Bip(2F); H-Aib-QGTFTSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-QGTFTSD-Bip(OMe)-Bip(2Me); H-(ACP)-QGTFTSD-Bip(OMe)-Bip(Pyr);H-(ACP)-QGTFTSD-Bip(OMe)-Bip(2F); H-(ACP)-QGTFTSD-Bip(OMe)-Bip(2CF₃);HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HSQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HAQGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-Aib-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-(ACP)-QGT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HSQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HSQGT-(□F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HAQGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-Aib-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-(ACP)-QGT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HS-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me); HS-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr);HS-(CNB)-GTFTSD-Bip(OMe)-Bip(2F); HS-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃);HA-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me); HA-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr);HA-(CNB)-GTFTSD-Bip(OMe)-Bip(2F); HA-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃);H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me);H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr);H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(2F);H-Aib-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(2Me);H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(Pyr);H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(2F);H-(ACP)-(CNB)-GTFTSD-Bip(OMe)-Bip(2CF₃);HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HS-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HA-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2Me);H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(Pyr);H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2F);H-Aib-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2CF₃);H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2Me);H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(Pyr);H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2F);H-(ACP)-(CNB)-GT-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2CF₃);HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HS-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HS-(CNB)-GT-(□F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HA-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-Aib-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-(ACP)-(CNB)-GT-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(2Me); HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr);HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(2F); HSQ-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃);HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(2Me); HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr);HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(2F); HAQ-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃);H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2Me);H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr);H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2F);H-Aib-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2Me);H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(Pyr);H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2F);H-(ACP)-Q-(ACP)-TFTSD-Bip(OMe)-Bip(2CF₃);HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HSQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HAQ-(ACP)-T-(α-OMe-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HAQ-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2Me);H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(Pyr);H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2F);H-Aib-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2CF₃);H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2Me);H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(Pyr);H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2F);H-(ACP)-Q-(ACP)-T-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2CF₃);HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HSQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HSQ-(ACP)-T-(□F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HAQ-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-Aib-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-(ACP)-Q-(ACP)-T-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HSQG-(PCA)-FTSD-Bip(OMe)-Bip(2Me); HSQG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr);HSQG-(PCA)-FTSD-Bip(OMe)-Bip(2F); HSQG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃);HAQG-(PCA)-FTSD-Bip(OMe)-Bip(2Me); HAQG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr);HAQG-(PCA)-FTSD-Bip(OMe)-Bip(2F); HAQG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃);H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(2Me);H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr);H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(2F);H-Aib-QG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(2Me);H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(Pyr);H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(2F);H-(ACP)-QG-(PCA)-FTSD-Bip(OMe)-Bip(2CF₃);HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HSQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2F);HAQG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2Me);H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(Pyr);H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2F);H-Aib-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2CF₃);H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2Me);H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(Pyr);H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2F);H-(ACP)-QG-(PCA)-(α-Me-2F-Phe)-TSD-Bip(OMe)- Bip(2CF₃);HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HSQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);HAQG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F);H-Aib-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃);H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2Me);H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(Pyr);H-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2F); andH-(ACP)-QG-(PCA)-(2F-Phe)-TSD-Bip(OMe)-Bip(2CF₃).


11. A pharmaceutical composition comprising a compound of formula (I) asclaimed in claim 1, and a pharmaceutically acceptable carrier(s).
 12. Amethod of treating a disease caused by hyperlipidaemia,hypercholesteremia, hyperglycemia, hyperinsulinemia, elevated bloodlevels of free fatty acids or glycerol, hypertriglyceridemia, woundhealing, impaired glucose tolerance, leptin resistance, insulinresistance or other diabetic complication comprising administering aneffective, non-toxic amount of compound of formula (I) as claimed inclaim 1 to a patient in need thereof.
 13. The method according to claim12, wherein the disease is type 2 diabetes, impaired glucose tolerance,dyslipidaemia, hypertension, atherosclerosis, hyperlipidaemia, coronaryartery disease, cardiovascular disorders and other diseases whereininsulin resistance is the underlying pathophysiological mechanism.
 14. Apharmaceutical composition comprising a compound as claimed in claim 10and a pharmaceutically acceptable carrier(s).
 15. A method of treating adisease caused by hyperlipidaemia, hypercholesteremia, hyperglycemia,hyperinsulinemia, elevated blood levels of free fatty acid or glycerol,hypertriglyceridemia, wound healing, impaired glucose tolerance, leptinresistance, insulin resistance or other diabetic complication comprisingadministering an effective, non-toxic amount of compound as defined inclaim 10 to a patient in need thereof.
 16. The method according to claim15, wherein the disease is type 2 diabetes, impaired glucose tolerance,dyslipidaemia, hypertension, atherosclerosis, hyperlipidaemia, coronaryartery disease, cardiovascular disorders and other diseases whereininsulin resistance is the underlying pathophysiological mechanism.
 17. Amethod of treating a disease caused by hyperlipidaemia,hypercholesteremia, hyperglycemia, hyperinsulinemia, elevated bloodlevels of free fatty acids or glycerol, hypertriglyceridemia, woundhealing, impaired glucose tolerance, leptin resistance, insulinresistance or other diabetic complication comprising administering aneffective, non-toxic amount of a composition according to claim 11 to apatient in need thereof.
 18. The method according to claim 17, whereinthe disease is type 2 diabetes, impaired glucose tolerance,dyslipidaemia, hypertension, atherosclerosis, hyperlipidaemia, coronaryartery disease, cardiovascular disorders and other diseases whereininsulin resistance is the underlying pathophysiological mechanism.
 19. Amethod of treating a disease caused by hyperlipidaemia,hypercholesteremia, hyperglycemia, hyperinsulinemia, elevated bloodlevels of free fatty acids or glycerol, hypertriglyceridemia, woundhealing, impaired glucose tolerance, leptin resistance, insulinresistance or other diabetic complication comprising administering aneffective, non-toxic amount of a composition as claimed in claim 14 to apatient in need thereof.
 20. The method according to claim 19, whereinthe disease is type 2 diabetes, impaired glucose tolerance,dyslipidaemia, hypertension, atherosclerosis, hyperlipidaemia, coronaryartery disease, cardiovascular disorders and other diseases whereininsulin resistance is the underlying pathophysiological mechanism.